Amino acid derivatives

ABSTRACT

Compounds of the formula    &lt;IMAGE&gt;  I  wherein R1, R2, R3, R4, R5, R6, and -N(R7)-CH(R8)(R9) are as claimed herein or their pharmaceutically acceptable acid addition salts inhibit proteases of viral origin and can be used as medicaments for the treatment of viral infections. They can be manufactured according to generally known procedures.

This is a division of application Ser. No. 07/916,812 filed Jul. 20,1992, now U.S. Pat. No. 5,446,161, which is a Divisional of Ser. No.07/362,621 filed Jun. 5, 1989, now U.S. Pat. No. 5,157,041.

SUMMARY OF THE INVENTION

The present invention is concerned with amino acid derivatives.

The amino acid derivatives provided by the present invention arecompounds of the general formula ##STR2##

wherein n stands for zero or 1; R¹ represents alkoxycarbonyl,aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl,heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl,monoaralkylcarbamoyl, cinnamoyl or α-aralkoxycarbonylaminoalkanoyl andR² represents hydrogen or R¹ and R² together with the nitrogen atom towhich they are attached represent a cyclic imide group of the formula##STR3## in which P and Q together represent an aromatic system; R³represents alkyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl,cyanoalkyl, alkylsulphinylalkyl, carbamoylalkyl oralkoxycarbonylalkylor, when n stands for zero, R³ can also represent alkylthioalkyl or,when n stands for 1, R³ can also represent alkylsulphonylalkyl; R⁴represents alkyl, cycloalkyl cycloalkylalkyl, aryl or aralkyl; R⁵represents hydrogen and R⁶ represents hydroxy or R⁵ and R⁶ togetherrepresent oxo; R⁷ and R⁸ together represent a trimethylene ortetramethylene group which is optionally substituted by hydroxy,alkoxycarbonylamino or acylamino or in which one --CH₂ -- group isreplaced by --NH--, --N(alkoxycarbonyl)-, --N(acyl)- or --S-- or whichcarries a fused cycloalkane, aromatic or heteroaromatic ring: and R⁹represents alkoxycarbonyl, monoalkylcarbamoyl, monoaralkylcarbamoyl,monoaryl- carbamoyl or a group of the formula ##STR4##

in which R¹⁰ and R¹¹ each represent alkyl; and pharmaceuticallyacceptable acid addition salts thereof.

DETAILED DESCRIPTION

The compounds of formula I and their pharmaceutically acceptable acidaddition salts are novel and possess valuable pharmacologicalproperties. In particular, they inhibit proteases of vital origin andcan be used in the prophylaxis or treatment of viral infections,particularly of infections caused by HIV and other retroid viruses.

Objects of the present invention are the compounds of formula I andtheir aforementioned salts per se and for use as therapeutically activesubstances, a process for the manufacture of said compounds and salts,intermediates used in said process, medicaments containing saidcompounds and salts, the use of said compounds and salts in the controlor prevention of illnesses, especially in the treatment or prophylaxisof vital infections, and the use of said compounds and salts for themanufacture of medicaments for the treatment or prophylaxis of vitalinfections.

As used in this Specification, the term "alkyl", alone or incombination, means a straight-chain or branched-chain alkyl groupcontaining a maximum of 8, preferably a maximum of 4, carbon atoms suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.butyl,tert.butyl, pentyl, hexyl and the like. The term "alkoxy", alone or incombination, means an alkyl ether group in which the term "alkyl" hasthe significance given earlier, such as methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy, sec.butoxy, tert.butoxy and the like.The term "cycloalkylalkyl" means an alkyl group as defined earlier whichis substituted by a cycloalkyl group containing 3-8, preferably 3-6,carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyland the like. The term "aryl", alone or in combination, means a phenylor naphthyl group which optionally carries one or more substituentsselected from alkyl, alkoxy, halogen, hydroxy, amino and the like, suchas phenyl, p-tolyl, 4-methoxyphenyl, 4-tert.butoxyphenyl,4-fluorophenyl, 4-chlorophenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyletc. The term "aralkyl", alone or in combination, means an alkyl groupas defined earlier in which one hydrogen atom is replaced by an arylgroup as defined earlier, such as benzyl, 2-phenylethyl and the like.The term "aralkoxycarbonyl", alone or in combination, means a group ofthe formula --C(O)--O-aralkyl in which the term "aralkyl" has thesignificance given earlier, such as benzyloxycarbonyl etc. The term"alkanoyl", alone or in combination, means an acyl group derived from analkanecarboxylic acid such as acetyl, propionyl, butyryl, valeryl,4-methylvaleryl etc. The term "cycloalkylcarbonyl" means an acyl groupderived from a monocyclic or bridged cycloalkanecarboxylic acid such ascyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl etc orfrom a benz-fused monocyclic cycloalkanecarboxylic acid which isoptionally substituted by, for example, alkanoylamino, such as1,2,3,4-tetrahydro-2-naphthoyl,2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl. The term "aralkanoyl" meansan acyl group derived from an aryl-substituted alkanecarboxylic acidsuch as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl),4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl),4-aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl etc. The term "aroyl"means an acyl group derived from an aromatic carboxylic acid; forexample an optionally substituted benzoic or naphthoic acid such asbenzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl,4-(benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl,6-carboxy-2-naphthoyl, 6-(benzyloxycarbonyl)-2-naphthoyl,3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl,3-(benzyloxyformamido)-2-naphthoyl etc. The heterocyclyl portion of aheterocyclylcarbonyl or heterocyclylalkyl group is a saturated,partially unsaturated or aromatic monocyclic, bicyclic ortricyclicheterocycle which contains one or more hetero atoms selectedfrom nitrogen, oxygen and sulphur, which is optionally substituted onone or more carbon atoms by halogen, alkyl, alkoxy, oxo etc and/or on asecondary nitrogen atom (i.e. --NH--) by alkyl, aralkoxycarbonyl,alkanoyl, phenyl or phenylalkyl or on a tertiary nitrogen atom (i.e.═N--) by oxido and which is attached via a carbon atom. Examples of suchheterocyclyl groups are pyrrolidinyl, piperidinyl, piperazinyl,morpholinyl, thiamorpholinyl, pyrrolyl, imidazolyl (e.g. imidazol-4-yl,1-benzyloxycarbonylimidazol-4-yl, etc), pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, furyl, thienyl, triazolyl, oxazolyl, thiazolyl, indolyl(e.g. 2-indolyl etc), quinolyl (e.g. 2-quinolyl, 3-quinolyl,1-oxido-2-quinolyl etc), isoquinolyl (e.g. 1-isoquinolyl, 3-isoquinolyletc), tetrahydroquinolyl (e.g. 1,2,3,4-tetrahydro-2-quinolyl etc),1,2,3,4-tetrahydroisoquinolyl (e.g. 1,2,3,4-tetrahydro-1-oxo-isoquinolyletc), quinoxalinyl, β-carbolinyl and the like. The term "halogen" meansfluorine, chlorine, bromine or iodine.

A cinnamoyl group denoted by R¹ can be unsubstituted or can carry on thephenyl ring one or more substituents selected from alkyl, alkoxy,halogen, nitro and the like.

The aromatic system denoted by P and Q together in formula (a) givenearlier can be monocyclic (e.g. 1,2-phenylene or thienylene) orpolycyclic (e.g. 1,2-naphthylene, 2,3-naphthylene, 1,8-naphthylene,2,3-anthrylene etc) and can be unsubstituted or substituted by one ormore substituents selected from alkyl, alkoxy, halogen and the like.

As mentioned earlier, a trimethylene or tetramethylene group denoted byR⁷ and R⁸ together can be optionally substituted by a hydroxy group oran alkoxycarbonylamino group (e.g. tert.butoxycarbonylamino) or anacylamino group (i.e. an alkanoylamino, cycloalkylcarbonylamino,aralkanoylamino or aroylamino group). Alternatively, one --CH₂ -- groupof a trimethylene or tetramethylene group denoted by R⁷ and R⁸ togethercan be replaced by --NH--, --N(alkoxycarbonyl)-, for example--N(tert.butoxycarbonyl)-, --N(acyl)- or --S--. When a trimethylene ortetramethylene group denoted by R⁷ and R⁸ together carries a fusedcycloalkane ring, this can be, for example, a fused cycloalkane ringcontaining 3-6 carbon atoms such as a fused cyclopentane, cyclohexane orlike ring and when the trimethylene or tetramethylene group carries afused aromatic or heteroaromatic ring, this can be, for example, a fusedbenzene, indole or thiophene ring which can be optionally substituted onone or more carbon atoms by halogen, alkyl, alkoxy etc. Thus,--N(R⁷)--CH(R⁸)(R⁹) can represent, for example, one of the followinggroups: ##STR5##

wherein R⁹ has the significance given earlier, R¹² represents hydrogen,hydroxy, alkoxycarbonylamino or acylamino, R¹³ represents hydrogen,alkoxycarbonyl or acyl, m stands for 1 or 2 and p stands for 1 or 2.

The pharmaceutically acceptable acid addition salts of the compounds offormula I are salts formed with inorganic acids, for example hydrohalicacids such as hydrochloric acid or hydrobromic acid, sulphuric acid,nitric acid, phosphoric acid etc, or with organic acids, for exampleacetic acid, citric acid, maleic acid, fumaric acid, tartaric acid,methanesulphonic acid, p-toluenesulphonic acid etc.

The compounds of formula I contain at least three asymmetric carbonatoms and are therefore present in the form of optically purediastereoisomers, mixtures of diastereoisomers, diastereoisomericracemates or mixtures of diastereoisomeric racemates. The presentinvention includes within its scope all of these forms.

One particular group of compounds of formula I comprises those in whichn stands for zero, R³ represents alkyl, cycloalkyl, aryl, aralkyl,heterocyclylalkyl, cyanoalkyl, alkylthioalkyl, carbamoylalkyl oralkoxycarbonylalkyl and R⁷ and R⁸ together represent a trimethylene ortetramethylene group in which one --CH₂ -group can be replaced by --NH--or --S-- or which can carry a fused cycloalkane, aromatic orheteroaromatic ring.

In the compounds of formula I hereinbefore, preferably R¹ representsalkoxycarbonyl, aralkoxycarbonyl, alkanoyl, cycloalkylcarbonyl,aralkanoyl, aroyl, hererocyclylcarbonyl orα-aralkoxycarbonylamino-alkanoyl, especially benzyloxycarbonyl,2-naphthoyl, 1-hydroxy-2-naphthoyl, 3-hydroxy-2-naphthoyl,3-benzyloxy-2-naphthoyl, 2-quinolylcarbonyl or 3-quinolylcarbonyl, andR² represents hydrogen. R³ preferably represents alkyl, cyanoalkyl,alkylthioalkyl or carbamoylalkyl, especially cyanomethyl,methylthiomethyl or carbamoylmethyl. R⁴ preferably represents aralkyl,especially benzyl. Preferably, R⁵ represents hydrogen and R⁶ representshydroxy. Preferably, --N(R⁷)--CH(R⁸)(R⁹) represents one of the groups offormulae (c) to (i) hereinbefore, especially a group of formula (c) inwhich R¹² represents hydrogen and m stands for 2 or R¹² representstert.butoxycarbonylamino and m stands for 1, a group of formula (d) inwhich R¹³ represents tert.butoxycarbonyl, a group of formula (e) inwhich m stands for 1, a group of formula (f) in which m and p both standfor 1, or a group of formula (g), (i) or (j). With respect to R⁹, thispreferably represents alkoxycarbonyl especially tert.butoxycarbonyl,monoalkylcarbamoyl, especially isobutylcarbamoyl or tert.butylcarbamoyl,or a group of formula (b), especially one in which R¹⁰ representssec.butyl and R¹¹ represents isobutyl.

From the foregoing it will be appreciated that particularly preferredcompounds of formula I are those in which R¹ representsbenzyloxycarbonyl, 2-naphthoyl, 1-hydroxy-2-naphthoyl,3-hydroxy-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 2-quinolylcarbonyl or3-quinolylcarbonyl and R² represents hydrogen, R³ representscyanomethyl, methylthiomethyl or carbamoylmethyl, R⁴ represents benzyl,R⁵ represents hydrogen and R⁶ represents hydroxy and --N(R⁷)--CH(R⁸)(R⁹)represents a group of formula (c) hereinbefore in which R¹² representshydrogen and m stands for 2 or R¹² represents tert.butoxycarbonylaminoand m stands for 1, a group of formula (d) hereinbefore in which R¹³represents tert.butoxycarbonyl, a group of formula (e) hereinbefore inwhich m stands for 1, a group of formula (f) hereinbefore in which m andp both stand for 1 or a group of formula (g), (i) or (j) hereinbeforeand R⁹ represents tert.butoxycarbonyl, isobutylcarbamoyl,tert.butylcarbamoyl or a group of formula (b) in which R¹⁰ representssec.butyl and R represents isobutyl.

Especially preferred compounds of formula I are:

N² -[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]--N¹ -tert.butyl-L-prolinamide,

N² -[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]--N¹ -isobutyl-L-prolinamide,

N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-thiazolidinecarboxamide,

N-tert.butyl1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2(S)-piperidinecarboxamide,

1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamide,

1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,

2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide,

N-tert.butyl-3-[2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-4(R)-thiazolidinecarboxamide,

N¹ -tert.butyl-N²-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]-L-prolinamideN² -oxide,

1-[3(S)-[[N-(benzyloxycarbonyl)-3-cyano-L-alanyl]amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,

1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide,

1-[3(S)-[[N-(benzyloxycarbonyl)-3-cyano-L-alanyl]amino]-2(R)-hydroxy,4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide,

N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide,

1-[3(S)-[[N-(3-benzyloxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,

N-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2-piperidinecarboxamide1-oxide,

N-tert.butyl1-[3(S)-[[N-(3-hydroxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-2(S)-piperidinecarboxamide,

trans-2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy,4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamide,

4-(tert.butoxycarbonyl)-N-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2(Ror S)-piperazinecarboxamide,

N-tert.butyl-1-[2(R)-hydroxy-3(S)-[[N-(1-hydroxy-2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-2(S)-piperidinecarboxamide,

trans-N-tert.butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(4aR,8aS)-isoquinoline-3(S)-carboxamideand

N-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-cysteinyl]amino]butyl]-2(S)-piperidinecarboxamide.

The most preferred compounds of formula I are:

N-tert.Butyl-1-[2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-2(S)-piperidinecarboxamide,

N-tert.butyl-octahydro-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamideand

N-tert.butyl-1,2,3,4-tetrahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]pyrido[3,4-b]indole-1(Ror S)-carboxamide.

According to the process provided by the present invention, thecompounds of formula I hereinbefore and their pharmaceuticallyacceptable acid addition salts are manufactured by

(a) for the manufacture of a compound of formula I in which n stands forzero, reacting a compound of the general formula ##STR6##

wherein R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ have the significance given earlier,

with an acid of the general formula ##STR7##

wherein R¹, R² and R³ have the significance given earlier,

or a reactive derivative thereof, or

(b) for the manufacture of a compound of formula I in which n stands forzero, R⁵ represents hydrogen and R⁶ represents hydroxy, reducing acompound of formula I in which n stands for zero and R⁵ and R⁶ togetherrepresent oxo, or

(c) for the manufacture of a compound of formula I in which n stands forzero and R¹ represents alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl,heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, cinnamoyl orα-aralkoxycarbonylaminoalkanoyl and R² represents hydrogen or R¹ and R²together with the nitrogen atom to which they are attached represent acyclic imide group of formula (a) hereinbefore, reacting a compound ofthe general formula ##STR8##

wherein R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ have the significance givenearlier,

with an agent yielding an alkanoyl, cycloalkylcarbonyl, aralkanoyl,aroyl, heterocyclylcarbonyl, alkylsulphonyl, arylsulphonyl, cinnamoyl orα-aralkoxycarbonylaminoalkanoyl group or with an agent forming a cyclicimide group of formula (a) hereinbefore, or

(d) for the manufacture of a compound of formula I in which n stands forzero and R¹ represents monoaralkylcarbamoyl and R² represents hydrogen,reacting a compound of formula IV hereinbefore with a compound of thegeneral formula

    R.sup.1' --N═C═O                                   V

wherein R^(1') represents aralkyl, or

(e) for the manufacture of a compound of formula I in which R³represents alkylsulphinylalkyl and n stands for zero, oxidizing acompound of formula I in which R³ represents alkylthioalkyl and n standsfor zero, or

(f) for the manufacture of a compound of formula I in which n stands for1, oxidizing a compound of formula I in which n stands for zero, or

(g) for the manufacture of a compound of formula I in which n stands for1 and R¹ represents an aromatic N-heterocyclylcarbonyl N-oxide group andR² represents hydrogen, oxidizing a compound of formula I in which nstands for 1 and R¹ represents an aromatic N-heterocyclylcarbonyl groupand R² represents hydrogen, or

(h) for the manufacture of a compound of formula I in which n strandsfor 1 and R³ represents alkylsulphonylalkyl, oxidizing a compound offormula I in which n stands for 1 and R³ represents alkylsulphinylalkyl,or

(i) for the manufacture of a compound of formula I in which R¹represents carboxy-substituted aroyl, hydroxy-substituted aroyl orhydrocinnamoyl and R² represents hydrogen, catalytically hydrogenating acompound of formula I in which R¹ representsbenzyloxycarbonyl-substituted aroyl, benzyloxy-substituted aroyl orcinnamoyl and R² represents hydrogen, or

(j) for the manufacture of a compound of formula I in which R³represents imidazol-4-yl and/or R⁴ represents hydroxy-substituted arylor hydroxy-substituted aralkyl and/or R⁷ and R⁸ together represent atrimethylene or tetramethylene group in which one --CH₂ -- group isreplaced by --NH--, treating a compound of formula I in which R³represents 1-(benzyloxycarbonyl)-imidazol-4-yl and/or R⁴ representstert.butoxy-substituted aryl or tert.butoxy-substituted aralkyl and/orR⁷ and R⁸ together represent a trimethylene or tetramethylene group inwhich one --CH₂ -- group is replaced by --N(tert.butoxycarbonyl)- with astrong acid, or

(k) for the manufacture of a compound of formula I in which R⁷ and R⁸together represent a trimethylene or tetramethylene group which issubstituted by acylamino or in which one --CH₂ -- group is replaced by--N(acyl)-, acylating a compound of the general formula ##STR9##

wherein n, R¹, R², R³, R⁴, R⁵, R⁶ and R⁹ have the significance givenearlier and R^(7') and R^(8') together represent a trimethylene ortetramethylene group which is substituted by amino or in which one --CH₂-- group is replaced by --NH--, and/or

(l) if desired, separating a mixture of diastereoisomeric racemates intothe diastereoisomeric racemates or optically pure diastereoisomers,and/or

(m) if desired, separating a mixture of diastereoisomers into theoptically pure diastereoisomers, and/or

(n) if desired, converting a compound of formula I obtained into apharmaceutically acceptable acid addition salt.

The reaction of a compound of formula II with an acid of formula III inaccordance with embodiment (a) of the process can be carried out inaccordance with methods known per se in peptide chemistry. Thus, when anacid of formula III is used, the reaction is preferably carried out inthe presence of a condensation agent such as hydroxybenzotriazole anddicyclohexylcarbodiimide. This reaction is conveniently carried out inan inert organic solvent such as an ether (e.g. diethyl ether,tetrahydrofuran etc) or dimethylformamide at a low temperature, suitablyat about -10° C. to +5° C. and especially at about 0° C. Suitablereactive derivatives of acids of formula III which can be used are, forexample, the corresponding acid halides (e.g. acid chlorides), acidanhydrides, mixed anhydrides, activated esters etc. When a reactivederivative is used, the reaction is conveniently carried out in an inertorganic solvent such as a halogenated aliphatic hydrocarbon (e.g.dichloromethane etc) or an ether (e.g. diethyl ether, tetrahydrofuranetc) and, where appropriate, in the presence or an organic base (e.g.N-ethylmorpholine, diisopropylethylamine etc) at a low temperature,suitably at about -10° C. to +5° C. and especially at about 0° C.

The reduction of a compound of formula I in which R⁵ and R⁶ togetherrepresent oxo in accordance with embodiment (b) of the process can becarried out according to methods known per se for the reduction of acarbonyl group to a hydroxy group. Thus, for example, the reduction canbe carried out using a complex metal hydride such as an alkali metalborohydride, especially sodium borohydride, in an appropriate organicsolvent such as an alkanol (e.g. methanol, ethanol, propanol,isopropanol etc). Conveniently, the reduction is carried out at aboutroom temperature.

In accordance with embodiment (c) of the process, suitable agents whichyield an alkanoyl, cycloalkylcarbonyl, aralkanoyl, aroyl,heterocyclylcarbonyl, alkylsulphonyl, cinnamoyl orα-aralkoxycarbonylaminoalkanoyl group are the corresponding acids orreactive derivatives thereof such as the corresponding acid halides(e.g. acid chlorides), acid anhydrides, mixed anhydrides, activatedesters etc and suitable agents which form a cyclic imide group offormula (a) hereinbefore are compounds of the formulaHOOC--P--Q--COOAralkyl in which P and Q have the significance givenearlier. The reaction of a compound of formula IV with theaforementioned agents is carried out in the same manner as thatdescribed earlier in connection with embodiment (a) of the process. Inthe reaction of a compound of formula IV with a compound of the formulaHOOC--P--Q--COOAralkyl, the initially formed reaction productspontaneously loses a molecule of aralkanol (HO-Aralkyl) with theformation of the cyclic imide group.

The reaction of a compound of formula IV with a compound of formula V inaccordance with embodiment (d) of the process can be carried out in amanner known per se. Thus, the reaction is conveniently carried out inan inert organic solvent such as a halogenated aliphatic hydrocarbon(e.g. dichloromethane etc) at a temperature between about 0° C. and roomtemperature, preferably at room temperature.

The oxidation in accordance with embodiments (e), (f), (g) and (h) ofthe process can be carried out according to known procedures. Theoxidation is preferably carried out using an organic peracid such asperacetic acid, perbenzoic acid, a haloperbenzoic acid such asm-chloroperbenzoic acid, perphthalic acid or the like, although it canalso be carried out using hydrogen peroxide. The oxidation isconveniently carried out in the presence of an organic solvent which isinert under the reaction conditions, for example an alkanol such asmethanol, ethanol etc, a halogenated hydrocarbon such as methylenechloride etc, and the like. The oxidation can be carried out within awide temperature range, for example a range between about -70° C. andabout room temperature.

The catalytic hydrogenation in accordance with embodiment (i) of theprocess can be carried out in a known manner. Conveniently, thecatalytic hydrogenation is carried out in the presence of a noble metalcatalyst, preferably a palladium catalyst such as palladium-on-carbon,and in an inert organic solvent (e.g. an alkanol such as ethanol,isopropanol etc) at about room temperature and under atmosphericpressure. When a compound of formula I in which R¹ representsnitro-substituted cinnamoyl and R² represents hydrogen is catalyticallyhydrogenated according to this embodiment, then there is obtained acompound of formula I in which R¹ represents amino-substitutedhydrocinnamoyl and R² represents hydrogen.

Embodiment (j) of the process can be carried out using a stronginorganic acid, for example a hydrohalic acid such as hydrogen chlorideor hydrogen bromide, or a strong organic acid, for example a halogenatedalkanecarboxylic acid such as trifluoroacetic acid and the like. Thisembodiment can be carried out according to known procedures; for examplein the presence or absence of an inert organic solvent (e.g. analkanecarboxylic acid ester such as ethyl acetate etc) and at atemperature between about 0° C. and about room temperature, preferablyat about room temperature.

The acylation of a compound of formula VI in accordance with embodiment(k) of the process can be carried out according to methods known per se.Conveniently, the acylation is carried out using an acyl halide such asan acyl chloride or bromide in the presence of an inert organic solventsuch as dimethylformamide etc and at a temperature between about 0° C.and room temperature. In place of an acid halide there can, of course,also be used a different reactive acid derivative such as an acidanhydride or the like.

The optional separations in accordance with embodiments (l) and (m) ofthe process can be effected according to conventional methods; forexample, by column chromatography, thin-layer chromatography, highpressure liquid chromatography etc.

The conversion of a compound of formula I into a pharmaceuticallyacceptable acid addition salt in accordance with embodiment (n) of theprocess can be carried out by treating such a compound in a conventionalmanner with an inorganic acid, for example a hydrohalic acid such ashydrochloric acid or hydrobromic acid, sulphuric acid, nitric acid,phosphoric acid etc, or with an organic acid such as acetic acid, citricacid, maleic acid, fumaric acid, tartaric acid, methanesulphonic acid,p-toluenesulphonic acid etc.

The compounds of formula II which are used as starting materials inembodiment (a) of the process are novel and also form an object of thepresent invention.

The compounds of formula II can be prepared, for example, by reacting acompound of the general formula ##STR10##

wherein R⁴ has the significance given earlier, R¹⁴ represents anamino-protecting group (e.g. tert.butoxycarbonyl or benzyloxycarbonyl)and X represents a chlorine or bromine atom, with a compound of thegeneral formula ##STR11##

wherein R⁷, R⁸ and R⁹ have the significance given earlier,

and either cleaving off the group R¹⁴ from the resulting compound of thegeneral formula ##STR12##

wherein R⁴, R⁷, R⁸, R⁹ and R¹⁴ have the significance given earlier,

to give a compound of formula II in which R⁵ and R⁶ together representoxo or reducing the compound of formula IX and cleaving off the groupR¹⁴ from the resulting compound of the general formula ##STR13##

wherein R⁴, R⁷, R⁸, R⁹ and R¹⁴ have the significance given earlier,

to give a compound of formula II in which R⁵ represents hydrogen and R⁶represents hydroxy.

The reaction of a compound of formula VII, preferably one in which R¹⁴represents benzyloxycarbonyl, with a compound of formula VIII can becarried out in a known manner; for example, in an inert organic solventsuch as a halogenated aliphatic hydrocarbon (e.g. dichloromethane etc)and in the presence of a base (e.g. a trialkylamine such astriethylamine etc), conveniently at about room temperature.

The cleavage of the group R¹⁴ from a compound of formula IX can also becarried out in a known manner: for example, using a strong inorganicacid such as a hydrohalic acid or a strong organic acid (e.g.trifluoroacetic acid etc), conveniently at about 0° C. to about roomtemperature. Alternatively, a hydrogenolytically-cleavableamino-protecting group R¹⁴ can be cleaved off using hydrogen in thepresence of a noble-metal catalyst (e.g. a palladium catalyst such aspalladium-on-carbon) in an organic solvent or solvent mixture which isinert under the reaction conditions (e.g. an alkanol such as ethanol,isopropanol etc, an alkanecarboxylic acid ester such as ethyl acetate,etc) and conveniently at about room temperature.

The reduction of a compound of formula IX to give a compound of formulaX can be carried out as described earlier in connection with thereduction of a compound of formula I in which n stands for zero and R⁵and R⁶ together represent oxo in accordance with embodiment (b) of theprocess of the invention.

The cleavage of the group R¹⁴ from a compound of formula X can becarried out in a manner analogous to that described earlier inconnection with the cleavage of the group R¹⁴ from a compound of formulaIX.

A further method for the preparation of compounds of formula II in whichR⁵ represents hydrogen and R⁶ represents hydroxy comprises firstlyreacting a compound of the general formula ##STR14##

wherein R⁴ and R¹⁴ have the significance given earlier,

with a compound of formula VIII hereinbefore, conveniently in an inertorganic solvent such as an alkanol (e.g. methanol etc),dimethylformamide or the like and at an elevated temperature,conveniently at about 60° C. to about 120° C., and then cleaving off thegroup R¹⁴ in the reaction product (a compound of formula X hereinbefore)as described earlier.

The compounds of formula IV which are used as starting materials inembodiments (c) and (d) of the process are novel and form a furtherobject of the present invention.

The compounds of formula IV can be prepared, for example, by cleavingoff the benzyloxycarbonyl or tert.butoxycarbonyl group R¹ from acompound of formula I in which n stands for zero, R¹ representsbenzyloxycarbonyl or tert.butoxycarbonyl and R² represents hydrogen.This cleavage is carried out in a manner analogous to that describedearlier in connection with the cleavage of the group R¹⁴ from a compoundof formula IX.

The compounds of formula VI wherein R^(7') and R^(8') together representa trimethylene or tetramethylene group in which one --CH₂ -- group isreplaced by --NH--, which are used as starting materials in embodiment(k) of the process, are a sub-group of compounds of formula I. Thecompounds of formula VI wherein R^(7') and R^(8') together represent atrimethylene or tetramethylene group which is substituted by amino,which are also used as starting materials in embodiment (k) of theprocess, are novel and also form an object of the present invention.They can be prepared, for example, by cleaving off the alkoxycarbonylgroup from a compound of formula I in which R⁷ and R⁸ together representa trimethylene or tetramethylene group which is substituted byalkoxycarbonylamino. The cleavage can be carried out according toconventional procedures; for example, by treatment with an acid such asa hydrogen halide (e.g. hydrogen chloride) in an inert organic solvent(e.g. an alkanecarboxylic acid ester such as ethyl acetate etc).

The starting materials of formula III and their reactive derivatives,the starting materials of formula V as well as the compounds of formulaeVII, VIII and XI hereinbefore, insofar as they are not known compoundsor analogues of known compounds, can be prepared in a similar manner tothe known compounds or as described in the Examples hereinafter or inanalogy thereto. Moreover, the agents used in embodiment (c) of theprocess are generally known compounds.

As mentioned earlier, the compounds of formula I and theirpharmaceutically acceptable acid addition salts inhibit proteases ofvital origin and are useful in the treatment or prophylaxis of viralinfections, particularly of infections caused by HIV and other retroidviruses.

The in vitro inhibition of HIV protease by the compounds provided by thepresent invention can be demonstrated by means of the following test:

HIV protease was expressed in E. coli and partially purified fromsoluble extracts of the bacterium by ammonium sulphate fractionation(0-30%), Protease activity was assayed using the protected hexapeptidesuccinyl-Ser-Leu-Asn-Tyr-Pro-Ile isobutylamide as the substrate.Cleavage of the substrate was quantified by measuring the production ofH-Pro-Ile isobutylamide by the spectrophotometric assay of N-terminalproline,

1.25 mM of substrate were dissolved in 125 mM of citrate buffer (pH 5.5)containing 0.125 mg/ml of Tween 20, 10 μl of a solution of variousconcentrations of the test compound (dissolved in methanol or dimethylsulphoxide and diluted with water containing 0.1% Tween 20) and 10 μl ofprotease were added to 80 μl of the above buffered substrate. Digestionwas carried out at 37° C. for a fixed period of time and was terminatedby the addition of 1 ml of colour reagent [30 μg/ml of isatin and 1.5mg/ml of 2-(4-chlorobenzoyl)benzoic acid in 10% acetone in ethanol(vol./vol.)]. The solution was heated in a water bath and then thepigmented residues were re-dissolved in 1 ml of 1% pyrogallol in 33%water in acetone (wt./vol./vol.). The optical density of the solutionwas measured spectrophotometrically at 599 nm. The formation ofH-Pro-Ile isobutylamide in the presence of the test compound wascompared with controls and the concentration of test compound requiredto give 50% inhibition (I₅₀) was determined by means of a graph plottedfrom the various concentrations of test compound used.

The results obtained in the foregoing test using representativecompounds of formula I as the test compound are compiled in thefollowing Table.

                  TABLE                                                           ______________________________________                                               Compound                                                                              I.sub.50  (μM)                                              ______________________________________                                               A       0.87                                                                  B       0.15                                                                  C       0.3                                                                   D       0.13                                                                  E       0.75                                                                  F       0.75                                                                  G       0.08                                                                  H       0.01                                                                  I       0.085                                                          ______________________________________                                        Compound A:                                                                             N-[N-[[N-(Benzyloxycarbonyl)-L-asparaginyl]-                                  L-phenylalanyl]methyl]-L-proline                                              tert.butyl ester.                                                   Compound B:                                                                             N-[3(S)-[[N-(Benzyloxycarbonyl)-L-asparaginyl]-                               amino]-2(R)-hydroxy-4-phenylbutyl]-                                           L-proline tert.butyl ester.                                         Compound C:                                                                             N-[3(S)-[[N-(Benzyloxycarbonyl)-L-asparaginyl]-                               amino]-2(S)-hydroxy-4-phenylbutyl]-                                           L-proline tert.butyl ester.                                         Compound D:                                                                             N.sup.2 -[N-[3(S)-[[N-(Benzyloxycarbonyl)-                                    L-asparaginyl]amino]-2(R and S)-hydroxy-4-                                    phenylbutyl]-L-prolyl]-N.sup.1 -isobutyl-L-                                   isoleucinamide (isomer 1; Example 13).                              Compound E:                                                                             N.sup.2 -[N-[3(S)-[[N-[N-                                                     (Benzyloxycarbonyl)-L-leucyl]-                                                L-asparaginyl]amino]-2(R and S)-hydroxy-4-                                    phenylbutyl]-L-prolyl]-N.sup.1 -isobutyl-L-                                   isoleucinamide (isomer 1: Example 14).                              Compound F:                                                                             N.sup.2 -[3(S)-[[N-(Benzyloxycarbonyl)-L-asparaginyl]-                        amino]-2(R and S)-hydroxy-4-phenylbutyl]-                                     N.sup.1 -isopentyl-L-prolinamide                                              (isomer 2; Example 17).                                             Compound G:                                                                             N.sup.2 -[3(S)-[[N-(Benzyloxycarbonyl)-                                       L-asparaginyl]amino]-2(R or S)-hydroxy-4-                                     phenylbutyl]-N.sup.1 -isobutyl-L-prolinamide                                  (isomer 2; Example 21).                                             Compound H:                                                                             N-[2(R)-Hydroxy-3(S)-[[N-(2-naphthoyl)-L-                                     asparaginyl]amino]-4-phenylbutyl]-L-proline                                   tert.butyl ester.                                                   Compound I:                                                                             2-[3(S)-[[N-(Benzyloxycarbonyl)-L-asparaginyl]-                               amino]-2(R)-hydroxy-4-phenyl-butyl]-                                          N-tert.butyl-1,2,3,4-tetrahydro-3(R,S)-                                       isoquinolinecarboxamide.                                        

The compounds of formula I and their pharmaceutically acceptable acidaddition salts can be used as medicaments (e.g. in the form ofpharmaceutical preparations). The pharmaceutical preparations can beadministered enterally such as orally (e.g. in the form of tablets,coated tablets, dragees, hard and soft gelatine capsules, solutions,emulsions or suspensions), nasally (e.g. in the form of nasal sprays) orrectally (e.g. in the form of suppositories). However, theadministration can also be effected parenterally such as intramuscularlyor intravenously (e.g. in the form of injection solutions).

For the manufacture of tablets, coated tablets, dragees and hardgelatine capsules the compounds of formula I and their pharmaceuticallyacceptable acid addition salts can be processed with pharmaceuticallyinert, inorganic or organic excipients. Lactose, maize starch orderivatives thereof, talc, stearic acid or its salts etc can be use, forexample, as such excipients for tablets, dragees and hard gelatinecapsules.

Suitable excipients for soft gelatine capsules are, for example,vegetable oils, waxes, fats, semi-solid and liquid polyols etc.

Suitable excipients for the manufacture of solutions and syrups are, forexample, water, polyols, saccharose, invert sugar, glucose etc.

Suitable excipients for injection solutions are, for example, water,alcohols, polyols, glycerol, vegetable oils etc.

Suitable excipients for suppositories are, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols etc.

Moreover, the pharmaceutical preparations can contain preserving agents,solubilizers, viscosity-increasing substances, stabilizing agents,wetting agents, emulsifying agents, sweetening agents, colouring agents,flavouring agents, salts for varying the osmotic pressure, buffers,coating agents or antioxidants. They can also contain still othertherapeutically valuable substances.

In accordance with the invention the compounds of formula I and theirpharmaceutically acceptable acid addition salts can be used in thetreatment or prophylaxis of vital infections, particularly of retrovitalinfections. The dosage can vary within wide limits and will, of course,be fitted to the individual requirements in each particular case. Ingeneral, in the case of oral administration there should suffice a dailydosage of about 3 mg to about 3 g, preferably about 10 mg to about 1 g(e.g. approximately 300 mg per person), divided in preferably 1-3 unitdoses, which can, for example, be of the same amount. It will, however,be appreciated that the upper limit given above can be exceeded whenthis is found to be indicated.

The following Examples illustrate the present invention. The solventsystems referred to in these Examples are as follows:

System A: 5% methanol in chloroform

System B: 10% methanol in chloroform

System C: chloroform:methanol:acetic acid:water (120:15:3:2)

System D: chloroform:methanol:acetic acid:water (90:15:3:2)

System E: chloroform:methanol:acetic acid:water (60:18:2:3)

System F: chloroform:methanol:acetic acid:water (240:24:3:2)

System G: dichloromethane:methanol:acetic acid:water (120:15:3:2)

System H: diethyl ether:n-hexane:methanol (47.5:47.5:5)

System I: dichloromethane:methanol:acetic acid:water (60:18:2:3)

System J: dichloromethane:methanol:acetic acid:water (120:15:2:3).

EXAMPLE 1

0.5 g (1.07 mmol) ofN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester was dissolved in 50 ml of isopropanol and 5 ml of ethyl acetateand hydrogenated for 5 hours in the presence of 0.406 g (2.14 mmol) oftoluene-4-sulphonic acid and 50 mg of 5% palladium-on-carbon. Thecatalyst was removed by filtration and the filtrate was evaporated. Theresidue was taken up in 5 ml of dichloromethane and 5 ml ofdimethylformamide and cooled in an ice/salt bath. 0.285 g (1.07 mmol) ofN-(benzyloxycarbonyl)-L-asparagine in 25 ml of dimethylformamide wasadded, followed by 0.145 g (1.07 mmol) of hydroxybenzotriazole, 0.221 g(1.07 mmol) of dicyclohexylcarbodiimide and 0.246 g (2.14 mmol) ofN-ethylmorpholine. The mixture was left to stir overnight, the separateddicyclohexylurea was removed by filtration and the filtrate wasevaporated to dryness in a vacuum. The resulting dark brown gum waspartitioned between ethyl acetate and water. The organic phase waswashed in sequence with 5% sodium bicarbonate solution and saturatedsodium chloride solution and then dried over sodium sulphate. Thesolvent was removed by evaporation and the solid was chromatographed onsilica gel using 5% isopropanol in ethyl acetate for the elution. Therewas obtained 0.125 g ofN-[N-[[N-(benzyloxycarbonyl)-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester as a pale yellow solid.

Analysis for: C₃₁ H₄₀ N₄ O₇ [580.69]. Calculated: C, 64.12; H, 6.94; N,9.65% Found: C, 63.18; H, 6.79; N, 9.8%; ash 1.2% Ash-free: C, 63.91; H,6.87; N, 9.92%.

The N-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-prolinetert.butyl ester used as the starting material was prepared by stirring0.77 g of [N-(benzyloxycarbonyl)-L-phenylalanyl]methyl bromide with 0.35g of L-proline tert.butyl ester and 0.203 g of triethylamine in 15 ml ofdichloromethane at room temperature overnight. The solvent was removedby evaporation-and the crude product was chromatographed on silica gelusing chloroform for the elution to give 0.65 g ofN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester as a solid of melting point 98°-99° C.

EXAMPLE 2

1.5 g (3.22 mmol) ofN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester were hydrogenated in 10 ml of ethyl acetate and 5 ml ofisopropanol in the presence of 0.15 g of 5% palladium-on-carbon and1.223 g (6.44 mmol) of toluene-4-sulphonic acid and the product wascoupled with 1.24 g (3.27 mmol) ofN-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparagine in the presence of 0.442g (3.27 mmol) of hydroxybenzotriazole, 0.675 g (3.27 mmol) ofdicyclohexylcarbodiimide and 0.753 g (6.55 mmol) of N-ethylmorpholine ina an analogous manner to that described in Example 1. After working-upthere was obtained 0.34 g of a solid which was chromatographed on silicagel using 5% isopropanol in dichloromethane for the elution. There wereobtained 310 mg ofN-[[N-[N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester; MS: m/e 694 [M+H]⁺.

The N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparagine used as the startingmaterial was prepared as follows:

4.5 g (12.4 mmol) of N-(benzyloxycarbonyl)-L-leucine succinimide esterwere dissolved in 40 ml of dimethylformamide and the solution was cooledin an ice/salt bath. A solution of 1.64 g (12.4 mmol) of L-asparagine in3.1 ml (12.4 mmol) of 4M sodium hydroxide solution were added andsubsequently 2 g (24.8 mmol) of sodium bicarbonate were added. Themixture was stirred at room temperature for 18 hours. The solvent wasremoved by evaporation and the residue was taken up in 100 ml of water.The pH was adjusted to about 9.5 with 2M sodium hydroxide solution andthe mixture was extracted twice with 25 ml of diethyl ether each time.The pH of the aqueous solution was adjusted to 2.5 with 3M hydrochloricacid. The solid crystalline product which separated from the solutionwas filtered off, washed with diethyl ether and dried. There wereobtained 3.5 g of N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparagine.

Analysis for: C₁₈ H₂₅ N₃ O₆ (379.42). Calculated: C, 56.98; H, 6.64; N,11.07% Found: C, 56.76; H, 6.62; N, 11.05%.

EXAMPLE 3

0.71 g (1.52 mmol) ofN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester was hydrogenated in the presence of 0.58 g (3.05 mmol) oftoluene-4-sulphonic acid over 5% palladium-on-carbon and the product wascoupled with 0.35 g (1.52 mmol) of N-(4-methylvaleryl)-L-asparagine inan analogous manner to that described in Example 1. The crude productwas purified by chromatography on silica gel using isopropanol in ethylacetate (5%-8% gradient) for the elution. There was obtained 0.23 g ofN-[[N-[N-(4-methylvaleryl)-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester as a solid; MS: m/e 545 [M+H]⁺.

The N-(4-methylvaleryl)-L-asparagine used as the starting material wasprepared as follows:

5.35 g (25 mmol) of 4-methylvaleric acid succinimide ester weredissolved in 40 ml of dimethylformamide and the solution was added to asolution, cooled in ice, of 3.3 g (25 mmol) of L-asparagine in 6.25 ml(25 mmol) of 4M sodium hydroxide solution and 5 ml of dimethylformamide.5.5 g (65 mmol) of sodium bicarbonate were added and the mixture wasstirred at room temperature for 18 hours. The solvent was removed byevaporation and the residue was taken up in 100 ml of water. The pH wasadjusted to 3 with 4M hydrochloric acid. The solvent was then removed byevaporation and the residue was taken up in methanol. Insoluble materialwas removed by filtration and the solvent was removed by evaporation.The crude product was chromatographed on silica gel using System D forthe elution. There were obtained 900 mg ofN-(4-methylvaleryl)-L-asparagine as a solid: Rf (System D): 0.25.

EXAMPLE 4

In an analogous manner to that described in Example 1, but usingN-(tert.butoxycarbonyl)-L-asparagine in place ofN-(benzyloxycarbonyl)-L-asparagine there was obtainedN-[N-[[N-(tert.butoxycarbonyl)-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester in the form of a solid; MS: m/e 546 [M]⁺.

EXAMPLE 5

1.3 g (2.79 mmol) ofN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester was hydrogenated in a mixture of 10 ml of ethyl acetate and 10 mlof isopropyl alcohol in the presence of 0.39 g of 5% palladium-on-carbonand 1.06 g (5.58 mmol) of toluene-4-sulphonic acid and the product wascoupled with 0.62 g (2.78 mmol) of N-(benzyloxycarbonyl)-L-alanine inthe presence of 0.572 g (2.78 mmol) of dicyclohexylcarbodiimide, 0.375 g(2.78 mmol) of hydroxybenzotriazole and 0.64 g (5.57 mmol) ofN-ethylmorpholine in a manner analogous to that described in Example 1.After working-up and chromatography on silica gel using 3% isopropanolin dichloromethane for the elution there was obtained 0.55 g ofN-[[N-[N-(benzyloxycarbonyl)-L-alanyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester as a solid: MS: m/e 538 [M+H]⁺.

EXAMPLE 6

In a manner analogous to that described in Example 1, fromN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester and N-(benzyloxycarbonyl)-L-glutamine there was obtainedN-[[N-[N-(benzyloxycarbonyl)-L-glutaminyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester; MS: m/e 595 [M+H]⁺.

EXAMPLE 7

0.45 g (0.77 mmol) ofN-[N-[[N-(benzyloxycarbonyl)-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester, prepared as described in Example 1, was dissolved in10 ml of isopropanol and the solution was stirred at room temperaturefor 45 minutes in the presence of 0.075 g (1.98 mmol) of sodiumborohydride. The solvent was removed by evaporation and the residue wastaken up in 50 ml of ethyl acetate and washed in sequence with water andsaturated sodium chloride solution. After drying over sodium sulphateand evaporation there was obtained 0.43 g of a solid which waschromatographed on silica gel using 2% methanol in dichloromethane forthe elution. There were obtained 80 mg ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(S)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a solid; Rf (System B): 0.38; MS: m/e 583 [M+H]⁺.

Further elution of the column with 5% methanol in dichloromethaneyielded 70 mg ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a solid: Rf (System B): 0.23; MS: m/e 583 [M+H]⁺.

EXAMPLE 8

0.3 g (0.45 mmol) ofN-[[N-[N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester, prepared as described in Example 2, in 10 ml ofisopropanol was treated at room temperature with 40 mg (1.06 mmol) ofsodium borohydride. After 1 hour the mixture was worked-up as describedin Example 7. There was obtained 0.32 g of a solid which waschromatographed on silica gel using 3% methanol in dichloromethane forthe elution. There were obtained 90 mg ofN-[3(S)-[[N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester (isomer 1) as asolid; Rf (System A): 0.26: MS: m/e 696 [M+H]⁺.

Further elution of the column with 5% methanol in dichloromethaneyielded 70 mg ofN-[3(S)-[[N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester (isomer 2) as asolid; Rf (System A): 0.19; MS: m/e 696 [M+H]⁺.

EXAMPLE 9

0.18 g (0.33 mmol) ofN-[[N-[N-(4-methylvaleryl)-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester, prepared as described in Example 3, was reduced with30 mg (0.80 mmol) of sodium borohydride in 10 ml of isopropanol asdescribed in Example 7. The resulting two isomers of N-[2(R orS)-hydroxy-3(S)-[[N-(4-methylvaleryl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester were separated by chromatography on silica gel usingmethanol in chloroform (5%-8% gradient) for the elution. There wereobtained 35 mg of isomer 1 in the form of a solid; Rf (System B): 0.2;MS: m/e 547 [M+H]⁺, and 27 mg of isomer 2 in the form of a solid; Rf(System B): 0.15; MS: m/e 547 [M+H]⁺.

EXAMPLE 10

0.6 g (1.1 mmol) ofN-[N-[[N-(tert.butoxycarbonyl)-L-asparaginyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester, prepared as described in Example 4, was reduced with0.1 g (2.65 mmol) of sodium borohydride in 10 ml of isopropanol asdescribed in Example 7. The two isomers were separated by chromatographyon silica gel using System C for the elution. There were obtained 115 mgof isomer 1 ofN-[3(S)-[[N-(tert.butoxycarbonyl)-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester in the form of asolid; Rf (System D): 0.23; MS: m/e 549 [M+H]⁺, and 100 mg of isomer 2in the form of a solid; Rf (System D): 0.15; MS: m/e 549 [M+H]⁺.

EXAMPLE 11

0.45 g (0.84 mmol) ofN-[[N-[N-(benzyloxycarbonyl)-L-alanyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester, prepared as described in Example 5, was reduced in 15ml of isopropanol in the presence of 80 mg of sodium borohydride asdescribed in Example 7. After working-up the two isomers were separatedby chromatography on silica gel using System A for the elution. Therewere obtained 70 mg of isomer 1 ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-alanyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester in the form of agum; Rf (System A): 0.38; MS: m/e 540 [M+H]⁺, and 50 mg of isomer 2 inthe form of a gum; Rf (System A) 0.21; MS: m/e 540 [M+H]⁺.

EXAMPLE 12

0.55 g (0.93 mmol) ofN-[[N-[N-(benzyloxycarbonyl)-L-glutaminyl]-L-phenylalanyl]methyl]-L-prolinetert.butyl ester, prepared as described in Example 6, was reduced in 15ml of isopropanol in the presence of 90 mg of sodium borohydride asdescribed in Example 7. After working-up, the product waschromatographed on silica gel using 2% methanol in chloroform for theelution. There were obtained 60 mg of isomer 1 ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-glutaminyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester in the form of asolid; Rf (System B): 0.37; MS: m/e 597 [M+H]⁺, and 65 mg of isomer 2 inthe form of a solid; Rf (System B): 0.25; MS: m/e 597 [M+H]⁺.

EXAMPLE 13

3 g (5.2 mmol) of N² -[N-[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-L-prolyl]-N¹ -isobutyl-L-isoleucinamide weretaken up in 25 ml of ethyl acetate and 10 ml of isopropanol andhydrogenated over 0.3 g of 5% palladium-on-carbon for 5 hours in thepresence of 1.97 g (10.36 mmol) of toluene-4-sulphonic acid. Thecatalyst was filtered off and the filtrate was evaporated. The solidobtained was dried over phosphorus pentoxide under a high vacuum andcoupled with 1.48 g (5.57 mmol) of N-(benzyloxycarbonyl)-L-asparagine inan analogous manner to that described in Example 1. 3.3 g of crude N₂-[N-[3(S)-[[N-(benzyloxycarbonyl)-L-asparginyl]amino]-2(R andS)-hydroxy-4-phenylbutyl]-L-prolyl]-N¹ -isobutyl-L-isoleucinamide wereobtained as a solid. The two isomers were separated by chromatography onsilica gel using System F for the elution. There were obtained 90 mg ofisomer 1 in the form of a solid; Rf (System F): 0.23; MS: m/e 695[M+H]⁺, and 0.55 g of isomer 2 in the form of a solid; Rf (System F):0.11; MS: m/e 695 [M+H]⁺.

The N₂ -[N-[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-L-prolyl]-N¹ -isobutyl-L-isoleucinamide usedas the starting material was prepared as follows:

(i) A solution of 20.0 g of N-(benzyloxycarbonyl)-L-isoleucine and 9.6ml of N-ethylmorpholine in 400 ml of tetrahydrofuran was cooled to -20°C. and 9.8 ml of isobutyl chloroformate were added dropwise. The mixturewas stirred for 5 minutes and then 5.52 g of isobutylamine were addeddropwise. The mixture was stirred at -15° C. for 20 minutes and was thenallowed to warm to room temperature. 20 ml of water were added and thesolution was then evaporated to dryness. The residue was partitionedbetween 200 ml of water and 800 ml of ethyl acetate and the organicsolution was washed with 200 ml of 5% citric acid solution, 200 ml ofsaturated sodium bicarbonate solution and 200 ml of water, dried oversodium sulphate and evaporated to dryness. The residue was trituratedwith diethyl ether to give 18.2 g of N² -(benzyloxycarbonyl)-N¹-isobutyl-L-isoleucinamide which was used in the next step withoutfurther purification.

(ii) A solution of 18.0 g of the above product in 200 ml of ethanol washydrogenated over 1.0 g of 10% palladium-on-carbon for 5 hours. Thecatalyst was removed by filtration and the filtrate was evaporated togive 9.8 g of N¹ -isobutyl-L-isoleucinamide as a colourless oil whichwas used directly in the next step,

(iii) A solution of 17.3 g of N-(benzyloxycarbonyl)-L-prolinesuccinimide ester and 9.35 g of N¹ -isobutyl-L-isoleucinamide in 120 mlof tetrahydrofuran was stirred at room temperature overnight. Thesolvent was removed by evaporation and the residue was partitionedbetween 200 ml of ethyl acetate and 250 ml of 5% citric acid solution.The organic solution was dried over anhydrous sodium sulphate andevaporated to dryness. The residue was recrystallized from a mixture ofethyl acetate and n-hexane to give 13.6 g of N₂-[N-(benzyloxycarbonyl)-L-propyl]-N¹ -isobutyl-L-isoleucinamide as awhite solid of melting point 87°-88° C.

(iv) A solution of 13.6 g of the above product in 500 ml of ethanol washydrogenated over 10% palladium-on-carbon for 2 hours. The catalyst wasremoved by filtration and the filtrate was evaporated to give 9.10 g ofN₂ -L-prolyl-N¹ -isobutyl-L-isoleucinamide as a colourless oil which wasused without further purification.

(v) A solution of 8.86 g of [N-(benzyloxycarbonyl)-L-phenylalanyl]methylbromide 6.71 g of N² -L-prolyl-N¹ -isobutyl-L-isoleucinamide and 2.60 gof triethylamine in 400 ml of dichloromethane was stirred at roomtemperature overnight. The solvent was removed by evaporation and thecrude product was chromatographed on silica gel using ethyl acetate forthe elution. The resulting product was triturated with a mixture ofpetroleum ether (b.p. 40°-60° C.) and ethyl acetate to give 8.70 g of N²-[N-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-prolyl]-N¹-isobutyl-L-isoleucinamide as an off-white solid of m.p. 80°-81° C.

(vi) A solution of 3.5 g of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-prolyl]-N¹-isobutyl-L-isoleucinamide in 200 ml of ethanol was treated with 1.0 gof sodium borohydride in a manner analogous to that described in Example7 to give 7.1 g of N₂ -[N-[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-L-prolyl]-N¹ -isobutyl-L-isoleucinamide.

Analysis for: C₃₃ H₄₈ N₄ O₅. 0.5 H₂ O. Calculated: C, 67.21; H, 8.37; N,9.50% Found: C, 67.29; H, 8.31; N, 9.47%.

EXAMPLE 14

0.78 g (1.34 mmol) of N² -[N-[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-L-prolyl]-N¹ -isobutyl-L-isoleucinamide washydrogenated and the product was coupled with 0.53 g (1.39 mmol) ofN-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparagine in an analogous mannerto that described in Example 1. 1.1 g of crude N²-[N-[3(S)-[[N-[N-(benzyloxycarbonyl)-L-leucyl]-L-asparaginyl]amino]-2(Rand S)-hydroxy-4-phenylbutyl]-L-prolyl]-N¹ -isobutyl-L-isoleucinamidewere obtained as a solid. The two isomers were separated bychromatography on silica gel using System F for the elution. There wereobtained 60 mg of isomer 1 in the form of a solid; Rf (System C): 0.34;MS: m/e 808 [M]⁺, and 50 mg of isomer 2 in the form of a solid; Rf(System C) 0.24; MS: m/e 808 [M]⁺.

EXAMPLE 15

0.275 g (0.473 mmol) ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester was taken up in 10 ml of isopropanol and hydrogenatedfor 18 hours over 50 mg of 5% palladium-on-carbon in the presence of0.18 g (0.94 mmol) of toluene-4-sulphonic acid. The catalyst wasfiltered off and the filtrate was evaporated. The resulting solid wasdissolved in 10 ml of dichloromethane and the solution was cooled in anice-bath. 50 mg (0.49 mmol) of acetic anhydride were added, followed by0.10 g (0.99 mmol) of triethylamine and 0.2 ml of pyridine. The mixturewas stirred at room temperature for 1 hour. The solvent was removed byevaporation and the residue was partitioned between ethyl acetate andwater. The organic phase was washed with 5% sodium bicarbonate solutionand with saturated sodium chloride solution and then dried overanhydrous sodium sulphate. The solvent was removed by evaporation togive 35 mg ofN-[3(S)-[(N-acetyl-L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester; Rf (System E): 0.28; MS: m/e 491 [M+H]⁺.

EXAMPLE 16

1 g (2.14 mmol) of N-[3(S)-(benzyloxyformamido)-2-(R andS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester was taken up in 25ml of methanol and hydrogenated over 250 mg of 5% palladium-on-carbonfor 3 hours in the presence of 0.81 g (4.26 mmol) of toluene-4-sulphonicacid. The catalyst was filtered off and the filtrate was evaporated. Theresulting solid was dried over phosphorus pentoxide under a high vacuumand then dissolved in 5 ml of tetrahydrofuran.

0.566 g (2.134 mmol) of N-(benzyloxycarbonyl)-L-leucine was taken up in15 ml of tetrahydrofuran and cooled to -15° C. 0.245 g (2.134 mmol) ofN-ethylmorpholine and 0.291 g (2.134mmol) of isobutyl chloroformate wereadded. After 5 minutes the tetrahydrofuran solution prepared asdescribed in the previous paragraph was added, followed by 0.451 g (4.27mmol) of N-ethylmorpholine. The mixture was stirred at room temperaturefor 10 hours, the solvent was then removed by evaporation and theresidue was partitioned between ethyl acetate and water. The ethylacetate solution was washed with saturated sodium bicarbonate solutionand with saturated sodium chloride solution and then dried overanhydrous sodium sulphate. The solvent was removed under a vacuum togive 1.5 g of crude N-[3(S)-[[N-(benzyloxycarbonyl)-L-leucyl]amino]-2-(Rand S)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester as an oil. Thetwo isomers were separated by chromatography on silica gel using 50%n-hexane in ethyl acetate for the elution. There were obtained 180 mg ofisomer 1 of N-[3(S)-[[N-(benzyloxycarbonyl)-L-leucyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester as a crystallinematerial; Rf (System A): 0.34; MS: m/e 582 [M+H]⁺, and 70 mg of isomer 2as a crystalline material; Rf (System A): 0.28; MS: m/e 582 [M+H]⁺.

The N-[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-L-proline tert.butyl ester used as thestarting material was prepared as follows:

2 g (4.3 mmol) ofN-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-L-proline tert.butylester (prepared as described in Example 1) were dissolved in 25 ml ofisopropanol, 25 ml of ethanol and 25 ml of methanol and the solution wasstirred at room temperature for 4 hours in the presence of 0.4 g (10.7mmol) of sodium borohydride. The solvent was removed by evaporation andthe residue was partitioned between 50 ml of ethyl acetate and 25 ml ofwater. The organic phase was washed with saturated sodium chloridesolution and dried over anhydrous sodium sulphate. The solvent wasremoved by evaporation and there were obtained 2 g ofN-[3(S)-(benzyloxyformamido)-2(R and S)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a clear gum: Rf (System C): 0.68 and 0.57.

EXAMPLE 17

1.5 g (3.12 mmol) of N₂ -[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-N¹ -isopentyl-L-prolinamide were hydrogenatedin 25 ml of methanol in the presence of 1.19 g (6.24 mmol) oftoluene-4-sulphonic acid and 0.25 g of 5% palladium-on-carbon. After 3hours the catalyst was filtered off and the filtrate was evaporated togive 2.1 g of a solid. This solid was dried over phosphorus pentoxideunder a high vacuum and coupled with 0.8 g (3.12 mmol) ofN-(benzyloxycarbonyl)-L-asparagine in a manner analogous to thatdescribed in Example 1. There were obtained 1.45 g of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R andS)-hydroxy-4-phenylbutyl]-N¹ -isopentyl-L-prolinamide as a crude productwhich was chromatographed on silica gel using System C for the elution.There were obtained 60 mg of isomer 1 as a solid; Rf (System E): 0.58;MS: 596 [M+H]⁺, and 0.25 g of isomer 2 as a solid, Rf (System E): 0.41;MS: m/e 596 [M+H]⁺.

The N² -[3(S)-(benzyloxyformamido)-2(R and S)-hydroxy-4-phenylbutyl]-N¹-isopentyl-L-prolinamide used as the starting material was prepared asfollows:

(i) A solution of 15.0 g of N-(benzyloxycarbonyl)-L-proline succinimideester and 4.15 g of isopentylamine in 100 ml of tetrahydrofuran wasstirred at room temperature overnight. The solvent was removed byevaporation and the residue was taken up in 250 ml of ethyl acetate. Thesolution was washed with 250 ml of 5% citric acid solution, two 250 mlportions of saturated sodium bicarbonate solution and 150 ml ofsaturated sodium chloride solution, dried over anhydrous sodium sulphateand evaporated to dryness. The residue was recrystallized from a mixtureof ethyl acetate and n-hexane to give 11.1 g of N²-(benzyloxycarbonyl)-N¹ -isopentyl-L-prolinamide as a white solid ofmelting point 110°-112° C.

(ii) A solution of 5.73 g of N₂ -(benzyloxycarbonyl)-N¹-isopentyl-L-prolinamide in 600 ml of ethanol was hydrogenated over 0.8g of 10% palladium-on-carbon catalyst for 3.75 hours. The catalyst wasremoved by filtration and the filtrate was evaporated to give 3.4 g ofN¹ -isopentyl-L-prolinamide as an oil which was used without furtherpurification.

(iii) A solution of 3.4 g of N¹ -isopentyl-L-prolinamide, 6.76 g of[N-[benzyloxycarbonyl)-L-phenylalanyl]methyl bromide and 2.0 g oftriethylamine in 360 ml of dichloromethane was stirred at roomtemperature overnight. The crude product was isolated in a manneranalogous to that described in Example 1 and was recrystallized from amixture of ethyl acetate and n-hexane to give 3.3 g of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-N¹-isopentyl-L-prolinamide as a white solid of melting point 82°-84° C.

(iv) A solution of 0.96 g of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-N¹-isopentyl-L-prolinamide in 40 ml of ethanol was treated with 0.17 g ofsodium borohydride in a manner analogous to that described in Example 2.The crude product was recrystallized from a mixture of diethyl ether andn-hexane to give 0.65 g of N² -[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-N¹ -isopentyl-L-prolinamide as a white solidof melting point 73°-82° C.

EXAMPLE 18

0.58 g (1.24 mmol) of N₂ -[3(S)-(benzyloxyformamido)-2(R orS)-hydroxy-4-phenylbutyl]-N¹ -tert.butyl-L-prolinamide was dissolved in25 ml of methanol and hydrogenated for 5 hours at room temperature inthe presence of 0.472 g (2.48 mmol) of toluene-4-sulphonic acid and 0.1g of 5% palladium-on-carbon. The catalyst was filtered off and thefiltrate was evaporated. There was obtained 0.82 g of a solid which wasdried over phosphorus pentoxide under a high vacuum and coupled with0.322 g (1.21 mmol) of N-(benzyloxycarbonyl)-L-asparagine indimethylformamide in a manner analogous to that described in Example 1.After working-up in a manner analogous to that described in Example 10there was obtained 0.6 g of crude product which was chromatographed onsilica gel using System C for the elution. There were obtained 225 mg ofN₂ -[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-N¹ -tert.butyl-L-prolinamide as a solid; MS:m/e 582 [M+H]⁺.

The N² -[3(S)-(benzyloxyformamido)-2(R or S)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide used as the starting material was prepared asfollows:

(i) 5.2 g (15 mmol) of N-(benzyloxycarbonyl)-L-proline succinimide esterand 1.63 g (22.3 mmol) of tert.butylamine were taken up in 50 ml ofdichloromethane and the mixture was stirred at -8° C. for 1 hour andthen at room temperature for 18 hours. The solution was then washed with5% citric acid solution, saturated sodium bicarbonate solution andsaturated sodium chloride solution and dried over anhydrous sodiumsulphate. After evaporation the solid was crystallized from ethylacetate/n-hexane to give 3 g of N² -(benzyloxycarbonyl)-N¹-tert.butyl-L-prolinamide; MS: m/e 305 [M+H]⁺.

(ii) 2.5 g (8.22 mmol) of N₂ -(benzyloxycarbonyl)-N¹-tert.butyl-L-prolinamide in 25 ml of methanol were hydrogenated for 5hours over 0.5 g of 5% palladium-on-carbon. The catalyst was removed byfiltration and the solvent was removed by evaporation to give 1.4 g ofan oil which crystallized upon standing in a refrigerator for severalhours. 0.35 g (2.05 mmol) of this solid was added to a solution of 0.77g (2.05 mmol) of [N-(benzyloxycarbonyl)-L-phenylalanyl]methyl bromide in15 ml of dichloromethane and subsequently 0.207 g (2.05 mmol) oftriethylamine were added. The mixture was stirred at room temperaturefor 2 hours. The solvent was removed by evaporation and the residue wastaken up in 25 ml of ethyl acetate. The solid was filtered off anddiscarded. The mother liquor was washed with water, saturated sodiumbicarbonate solution and saturated sodium chloride solution and thendried over anhydrous sodium sulphate. After evaporation andcrystallization from ether/n-hexane there was obtained 0.65 g of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-N¹-tert.butyl-L-prolinamide in the form of a solid; MS: m/e 466 [M+H]⁺.

(iii) 1.25 g (2.7 mmol) of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-N¹-tert.butyl-L-prolinamide was taken up in 25 ml of isopropanol andstirred for 4 hours at room temperature in the presence of 0.255 g (6.7mmol) of sodium borohydride. The solvent was removed by evaporation andthe residue was partitioned between ethyl acetate and water. The organiclayer was washed with saturated sodium chloride solution, dried overanhydrous sodium sulphate and evaporated. There were obtained 1.2 g ofN² -[3(S)-(benzyloxyformamido)-2(R and S)-hydroxy-4-phenylbutyl]-N-tert.butyl-L-prolinamide which was chromatographed on silica gel usingSystem F for the elution. There were obtained 15 mg of isomer 1 as agum; Rf (System F): 0.38; MS: m/e 468 [M+H]⁺, and 0.225 g of isomer 2 asa gum; Rf (System F): 0.27; MS: m/e 468 [M+H]⁺.

EXAMPLE 19

A solution of 474 mg of N-[3(S)-(benzyloxyformamido)-4-cyclohexyl-2(R orS)-hydroxybutyl]-L-proline tert.butyl ester in 100 ml of ethanol washydrogenated over 50 mg of 10% palladium-on-carbon catalyst for 3 hours.The catalyst was removed by filtration and the filtrate was evaporatedto give 380 mg of an oil. This was coupled with 285 mg ofN-(benzyloxycarbonyl)-L-asparagine in a manner analogous to thatdescribed in Example 1. The crude product was chromatographed on silicagel using 7% methanol in dichloromethane for the elution to give 30 mgof N-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-4-cyclohexyl-2(Ror S)-hydroxybutyl]-L-proline tert.butyl ester as a white solid ofmelting point 135°-136° C. (isomer 1).

Similarly, 474 mg of the other isomer ofN-[3(S)-(benzyloxyformamido)-4-cyclohexyl-2(R orS)-hydroxybutyl]-L-proline tert.butyl ester were hydrogenated andcoupled with 285 mg of N-(benzyloxycarbonyl)-L-asparagine to give 90 mgof isomer 2, MS: m/e 589 [M+H]⁺.

The N-[3(S)-(benzyloxyformamido)-4-cyclohexyl-2-(R andS)-hydroxybutyl]-L-proline tert.butyl ester used as the startingmaterial was prepared as follows:

(i) A solution of 19.6 g of N-(benzyloxycarbonyl)-3-cyclohexyl-L-alaninein 60 ml of tetrahydrofuran was cooled to -10° C. and 8.9 ml ofN-ethylmorpholine were added. 11.6 ml of isobutyl chloroformate wereadded dropwise and the mixture was stirred at -10° C. for a further 15minutes. 250 ml of diethyl ether were added and the mixture wasfiltered. The filtrate was added to a cold solution of diazomethane indiethyl ether (prepared from 21.5 g ofN-methyl-N-nitroso-4-toluenesulphonamide) and the mixture was stirred atroom temperature for 1.5 hours. The solution was then washed with two200 ml portions of water and with 200 ml of saturated sodium bicarbonatesolution, dried over anhydrous sodium sulphate and evaporated to give6.6 g of benzyl [2-cyclohexyl-1(S)-(2-diazoacetyl)ethyl]carbamate as ayellow oil which was used without further purification.

(ii) A solution of 6.6 g of the above product in 60 ml of diethyl etherwas stirred at room temperature while hydrogen chloride was bubbledthrough the solution for 30 minutes. The solvent was removed byevaporation and the resulting oil was crystallized from a mixture ofdiethyl ether and n-hexane to give 5.8 g of[N-(benzyloxycarbonyl)-3-cyclohexyl-L-alanyl]methyl chloride which wasused without further purification.

(iii) A solution of 4.4 g of the above product in a mixture of 90 ml oftetrahydrofuran and 10 ml of water was cooled to 0° C. and 0.75 g ofsodium borohydride was added. The mixture was stirred at 0° C. for 1hour and then evaporated to dryness. The residue was taken up in 100 mlof dichloromethane and 100 ml of water, the aqueous layer was adjustedto pH 3 with concentrated hydrochloric acid and the organic layer wasseparated, dried over anhydrous sodium sulphate and evaporated. Theresidue was triturated with 200 ml of hot n-hexane and filtered off togive 2.65 g of 3(S)-(benzyloxyformamido)-1-chloro-4-cyclohexyl-2(R andS)-butanol as a white solid; MS: 339, 341 [M]⁺.

(iv) 14 ml of 0.71M ethanolic potassium hydroxide solution were added toa solution of 2.65 g of the above product in 60 ml of ethanol. Themixture was stirred at room temperature for 1 hour and then evaporatedto dryness. The residue was partitioned between 100 ml ofdichloromethane and 100 ml of water and the organic layer was dried overanhydrous sodium sulphate and evaporated. The residue waschromatographed on silica gel using 3% methanol in dichloromethane forthe elution. There were obtained 1.84 g of3(S)-(benzyloxyformamido)-4-cyclohexyl-1,2(R and S)-epoxybutane as anoil; MS: m/e 303 [M]⁺.

(v) A solution of 1.84 g of the above product and 1.21 g of L-prolinetert.butyl ester in 65 ml of methanol was heated under reflux for 24hours. The mixture was evaporated to dryness and the crude product waschromatographed on silica gel using 5% methanol in dichloromethane forthe elution. There was obtained 0.41 g of N₂-[3(S)-(benzyloxyformamido)-4-cyclohexyl-2(R orS)-hydroxybutyl]-L-proline tert.butyl ester (isomer 1) as a colourlessoil; NMR (CDCl₃, 250 MHz) δ0.78-1.0 (2H, m), 1.1-1.4 (5H, m), 1.43 (9H,s), 1.55-1.7 (5H, m) 1.8-1.95 (4H, m), 2.03-2.15 (1H, m), 2.3-2.4 (1H,m), 2.5 (1H, dd), 2.66 (1H, t), 3.16-3.26 (2H, m) 3.55-3.7 (2H, m), 4.08(1H, broad s), 5.02-5.15 (3H, m), 7.25-7.4 (5H, m).

Further elution with the same solvent system yielded 1.18 g of isomer 2as a colourless oil; NMR (CDCl₃, 300 MHz) δ0.75-0.85 (1H, m), 0.9-1.0(1H, m), 1.1-1.4 (5H, m), 1.43. (9H, s), 1.58-1.70 (5H, m),1.75-1.9 (5H,m), 2.05-2.18 (1H, m), 2.54-2.75 (3H,. m), 3.05-3.15 (2H, m), 3.45-3.52(1H, m), 3.7-3.8 (1H, m), 4.96-5.15 (3H, m), 7.3-7.38 (5H, m).

EXAMPLE 20

0.663 g (1.42 mmol) of N₂ -[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-N¹ -isobutyl-L-prolinamide was hydrogenated in30 ml of methanol in the presence of 0.539 g (2.84 mmol) oftoluene-4-sulphonic acid and 0.1 g of 5% palladium-on-carbon, and theproduct was then coupled with 0.378 g (1.42 mmol) ofN-(benzyloxycarbonyl)-L-asparagine and worked-up as described inExample 1. The crude product was subjected to flash chromatography onsilica gel using System F and there were obtained 80 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R orS)-hydroxy-4-phenylbutyl]-N¹ -isobutyl-L-prolinamide (isomer 1) as asolid; Rf (System E) 0.46; MS: m/e 582 [M+H]⁺.

The column was then eluted with System C to give 70 mg of isomer 2 as asolid; Rf (System E) 0.33; MS: m/e 582 [M+H]⁺.

The N₂ -[3(S)-(benzyloxyformamido)-2(R and S)-hydroxy-4-phenylbutyl]-N¹-isobutyl-L-prolinamide used as the starting material was prepared asfollows:

(i) N¹ -isobutyl-L-prolinamide was prepared as described in Example17(i) using isobutylamine in place of isopentylamine.

(ii) A solution of 1.5 g (3.98 mmol) of[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl bromide, 0.678 g (3.98mmol) of N¹ -isobutyl-L-prolinamide and 0.402 g (3.98 mmol) oftriethylamine in 30 ml of dichloromethane was stirred at roomtemperature for 4 hours. The solvent was removed by evaporation and 50ml of ethyl acetate were added. The resulting solid was removed byfiltration and discarded. The filtrate was washed with water, sodiumbicarbonate solution and saturated sodium chloride solution and thendried over anhydrous sodium sulphate. The solvent was removed byevaporation and the crude product was chromatographed on silica gelusing chloroform for the elution to give 1.6 g of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-N¹-isobutyl-L-prolinamide as a solid; MS: m/e 466 [M+H]⁺.

(iii) A solution of 0.7 g (1.5 mmol) of N₂-[[N-(benzyloxycarbonyl)-L-phenylalanyl]methyl]-N¹-isobutyl-L-prolinamide in 20 ml of ethanol was treated with 0.143 g(3.75 mmol) of sodium borohydride at room temperature for 2 hours. Thesolvent was removed by evaporation and the residue was partitionedbetween ethyl acetate and water. The ethyl acetate phase was washed withsaturated sodium chloride solution, dried over anhydrous sodium sulphateand evaporated to give 0.67 g of N₂ -[3(S)-(benzyloxyformamido)-2(R andS)-hydroxy-4-phenylbutyl]-N¹ -isobutyl-L-prolinamide as a solid; Rf(System A) 0.39 and 0.33; MS: m/e 467 [M]⁺.

EXAMPLE 21

A solution of 249 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 106 mg of 2-naphthoyl chloride and 72 mg ofdiisopropylethylamine in 10 ml of dry dichloromethane was stirred at 20°C. for 20 hours. The gelatinous reaction mixture was partitioned betweendichloromethane and water. The organic phase was evaporated and theresulting oil was crystallized from ethyl acetate/n-hexane to give 215mg ofN-[2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester, as a white solid; MS: m/e 603 [M+H]⁺.

The N-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester used as the starting material was prepared as follows:

A solution of 1.75 g ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester in 100 ml of ethanol was hydrogenated over 10%palladium-on-carbon for 64 hours. The catalyst was removed by filtrationand the filtrate was evaporated. The residual gum was partitionedbetween ethyl acetate and 2M hydrochloric acid. The aqueous phase wasmade basic with dilute sodium hydroxide solution and extracted withethyl acetate. The ethyl acetate extract was evaporated to give 1.14 gof N-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a white, solid foam; MS: m/e 449 [M+H]⁺.

EXAMPLE 22

In a manner analogous to that described in Example 21, from 223 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 95 mg of toluene-4-sulphonyl chloride and 65 mg ofdiisopropylethylamine there were obtained 170 mg ofN-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(p-toluenesulphonyl)-L-asparaginyl]amino]butyl]-L-prolinetert.butyl ester as a white solid (from ethylacetate/n-hexane): MS: m/e603 [M+H]⁺.

EXAMPLE 23

In a manner analogous to that described in Example 21, from 223 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 77 mg of phenylacetyl chloride and 65 mg ofdiisopropylethylamine there were obtained 190 mg ofN-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(phenylacetyl)-L-asparaginyl]amino]butyl]-L-prolinetert.butyl ester as a white solid (from ethyl acetate/n-hexane): MS: m/e567 [M+H]⁺.

EXAMPLE 24

In a manner analogous to that described in Example 21, from 249 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 94 mg of hydrocinnamoyl chloride and 72 mg ofdiisopropylethylamine there were obtained 214 mg ofN-[3(S)-[(N-hydrocinnamoyl-L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid (from ethyl acetate/n-hexane); MS: m/e581 [M+H]⁺.

EXAMPLE 25

In a manner analogous to that described in Example 21, from 270 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 114 mg of 1-naphthoyl chloride and 77 mg ofdiisopropylamine there were obtained, after chromatography on silica gelusing 10% methanol in dichloromethane for the elution, 130 mg ofN-[2(R)-hydroxy-3(S)-[[N-(1-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 603 [M+H]⁺.

EXAMPLE 26

In a manner analogous to that described in Example 21, from 270 mg ofN-[3(S)-[[n-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 100 mg of cinnamoyl chloride and 77 mg ofdiisopropylethylamine there were obtained, after chromatography onsilica gel using 10% methanol in dichloromethane for the elution, 99 mgofN-[3(S)-[(N-cinnamoyl-L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 579 [M+H]⁺.

EXAMPLE 27

A solution of 270 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester and 98 mg of 4-phenylbutyric acid in 10 ml of drytetrahydrofuran was cooled in an ice/salt mixture. 81 mg ofhydroxybenzotriazole, 69 mg of N-ethylmorpholine and 136 mg ofdicyclohexylcarbodiimide were added and the mixture was stirred for 64hours. The mixture was then diluted with ethyl acetate and filtered. Thefiltrate was washed with aqueous sodium bicarbonate solution and sodiumchloride solution. The solvent was removed by evaporation and theresidue was chromatographed on silica gel using System G for the elutionto give 79 mg ofN-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(4-phenylbutyryl)-L-asparaginyl]amino]butyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 595 [M+H]⁺.

EXAMPLE 28

In a manner analogous to that described in Example 27, from 249 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 104 mg of 2-naphthylacetic acid, 75 mg ofhydroxybenzotriazole, 64 mg of N-ethylmorpholine and 126 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using 10% methanol in dichloromethane for the elution, 62 mgofN-[2(R)-hydroxy-3(S)-[[N-[(2-naphthyl)acetyl]-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid: MS: m/e 617 [M+H]⁺.

EXAMPLE 29

In a manner analogous to that described in Example 27, from 270 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 108 mg of 4-methoxyhydrocinnamic acid, 81 mg ofhydroxybenzotriazole, 69 mg of N-ethylmorpholine and 136 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using System G for the elution, 113 mg ofN-[2(R)-hydroxy-3(S)-[[N-(4-methoxyhydrocinnamoyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 611 [M+H]⁺.

EXAMPLE 30

0.179 g of dicyclohexylcarbodiimide, 0.016 g of hydroxybenzotriazole and0.09 g of N-ethylmorpholine were added to a stirred solution of 0.238 gof N-[3(S)-amino-2(R or S)-hydroxy-5-methylhexyl]-L-proline tert.butylester and 0.210 g of N-(benzyloxycarbonyl)-L-asparagine in 10 ml oftetrahydrofuran at 0° C. The mixture was stirred for 16 hours and thenfiltered. The filtrate was partitioned between ethyl acetate and waterand the organic phase was then washed with sodium bicarbonate solutionand sodium chloride solution. The organic phase was evaporated and theresidue was chromatographed on silica gel using ethyl acetate/methanol(9:1) for the elution to give, after recrystallization from ethylacetate/n-hexane, 0.13 g ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R orS)-hydroxy-5-methylhexyl)]-L-proline tert.butyl ester as a white solid;MS: m/e 549 [M+H]⁺.

The N-[3(S)-amino-2(R or S)-hydroxy-5-methylhexyl]-L-proline tert.butylester used as the starting material was prepared as follows:

(i) A solution of 1.31 g of 3(S)-(benzyloxyformamido)-1,2(R orS)-epoxy-5-methylhexane and 0.855 g of proline tert.butyl ester in 10 mlof dimethylformamide was stirred at 100° C. for 16 hours and the solventwas then removed by evaporation. The residue was partitioned betweenethyl acetate and water. The organic phase was washed with sodiumchloride solution and evaporated. The residue was chromatographed onsilica gel using ethyl acetate/acetic acid/ethanol (4:1:1) for theelution. Material having a Rf of approximately 0.24 wasre-chromatographed on silica gel using System H for the elution to give0.358 g of N-[3(S)-(benzyloxyformamido)-2(R orS)-hydroxy-5-methylhexyl]-L-proline tert.butyl ester in the form of acolourless oil; MS: m/e 435 [M+H]⁺.

(ii) A solution of 0.35 g of N-[3(S)-(benzyloxyformamido)-2(R orS)-hydroxy-5-methylhexyl)]-L-proline tert.butyl ester in 20 ml ofethanol was hydrogenated over 10% palladium-on-carbon for 16 hours. Thecatalyst was removed by filtration and the filtrate was evaporated togive 0.238 g of N-[3(S)-amino-2(R or S)-hydroxy-5-methylhexyl]-L-prolinetert.butyl ester in the form of a colourless gum: MS: m/e 301 [M+H]⁺.

EXAMPLE 31

A solution of 0.28 g ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, and 0.083 g of benzyl isocyanate in 5 ml ofdichloromethane was stirred at 20° C. for 2 hours. The solvent was thenremoved by evaporation and the residue was triturated with diethyl etherto give 0.174 g ofN-[3(S)-[[N-(benzylcarbamoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 582 [M+H]⁺.

EXAMPLE 32

A solution of 0.28 g ofN-[3(S)-[[L-asparaginyl]-amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 0.124 g of 1-adamantanecarbonyl chloride and 0.081 gof diisopropylethylamine in 5 ml of dichloromethane was stirred at 20°C. for 8 hours. A further 0.124 g of 1-adamantanecarbonyl chloride and0.081 g of diisopropylethylamine were added and stirring was continuedat 20° C. for 16 hours. The solution was diluted with 20 ml ofdichloromethane and washed in sequence with water, sodium carbonatesolution, potassium bisulphate solution and sodium chloride solution.The organic solution was then evaporated and the residue was trituratedwith diethyl ether to give 0.2 g ofN-[3(S)-[[N-(1-adamantylcarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl-L-prolinetert.butyl ester as a white solid; MS: m/e 611 [M+H]⁺.

EXAMPLE 33

0.208 g of dicyclohexylcarbodiimide, 0.124 g of hydroxybenzotriazole and0.106 g of N-ethylmorpholine were added to a stirred solution of 0.315 gof N² -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -phenyl-L-prolinamideand 0.244 g of N-(benzyloxycarbonyl)-L-asparagine in 10 ml oftetrahydrofuran at 0° C. The mixture was stirred for 16 hours, thendiluted with ethyl acetate and filtered. The filtrate was washed withsodium bicarbonate solution and sodium chloride solution and the solventwas then removed by evaporation. The residue was chromatographed onsilica gel using dichloromethane/methanol (9:1) for the elution to give0.22 g of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-phenyl-L-prolinamide in the form of a white solid of melting point169°-171° C. (from methanol).

The N² -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -phenyl-L-prolinamideused as the starting material was prepared as follows:

(i) 3.5 g of sodium borohydride were added to an ice-cooled stirredsolution of 21 g of [N-(benzyloxycarbonyl)-L-phenylalanyl]methylchloride in 500 ml of aqueous tetrahydrofuran. Stirring was continuedfor 0.5 hour and the solvent was removed by evaporation. The residue waspartitioned between dichloromethane and water and then carefullyacidified with concentrated hydrochloric acid. After working-up theorganic phase gave a white solid which ran as two spots; Rf 0.4 and 0.5(5% methanol/chloroform). The solids were extracted with boiling hexaneuntil all of the higher running component had been extracted. Thecombined n-hexane extracts were evaporated and the residue wasre-extracted with boiling n-hexane to remove a small amount (0.5 g) ofthe lower running component. In this manner there were obtained 11.5 gof 3(S)-(benzyloxyformamido)-1-chloro-4-phenyl-2(S)-butanol of meltingpoint 151°-153° C. (from ethyl acetate/n-hexane).

(ii) A solution of 11.4 g of3(S)-(benzyloxyformamido)-1-chloro-4-phenyl-2(S)-butanol in 300 ml ofethanol containing 2.24 g of potassium hydroxide was left to stand for15 minutes. The solvent was removed by evaporation and the residue waspartitioned between water and dichloromethane. Working-up gave 10.1 g ofcrude 3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane as anoff-white solid which was used without further purification.

(iii) A solution of 0.594 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane and 0.38 g of N¹-phenyl-L-prolinamide in 5 ml of dimethylformamide was heated to 90° C.for 16 hours and then to 120° C. for 8 hours. The solvent was removed byevaporation and the residue was chromatographed on silica gel usingdiethyl ether/methanol (96:4) for the elution to give 0.48 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-phenyl-L-prolinamide in the form of a white solid of melting point133°-135° C. (from diethyl ether).

(iv) A solution of 0.45 g of N²-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-phenyl-L-prolinamide in 20 ml of ethanol was hydrogenated over 10%palladium-on-carbon for 4 hours to give 0.315 g of N²-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -phenyl-L-prolinamide in theform of a white solid; MS: m/e 354 [M+H]⁺.

EXAMPLE 34

In a manner analogous to that described in Example 33, from 0.987 mg of2-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydro-3(R,S)-isoquinolinecarboxamide,0.665 g of N-(benzyloxycarbonyl)-L-asparagine, 566 mg ofdicyclohexylcarbodiimide, 0.337 g of hydroxybenzotriazole and 0.287 g ofN-ethylmorpholine there was obtained 0.5 g of2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydro-3(R,S)-isoquinolinecarboxamideas a pale cream foam; MS: m/e 644 [M+H]⁺.

EXAMPLE 35

In a manner analogous to that described in Example 33, from 0.225 g ofdicyclohexylcarbodiimide, 0.135 g of hydroxybenzotriazole, 0.115 g ofN-ethylmorpholine, 0.38 g of N²-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -benzyl-L-prolinamide and0.266 g of N-(benzyloxycarbonyl)-L-asparagine there was obtained 0.056 gof N¹ -benzyl-N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinamideas a white solid; MS: m/e 616 [M+H]⁺.

EXAMPLE 36

A solution of 270 mg ofN-[3(S)-(benzyloxyformamido)-4-(4-fluorophenyl)-2(R)-hydroxybutyl]-L-prolinetert.butyl ester in 50 ml of ethanol was hydrogenated over 30 mg of 10%palladium-on-carbon for 2 hours. The catalyst was removed by filtrationand the filtrate was evaporated to give an oil which was coupled with158 mg of N-(benzyloxycarbonyl)-L-asparagine in a manner analogous tothat described in Example 1. The crude product was chromatographed onsilica gel using 10% methanol in dichloromethane for the elution. Therewere obtained 120 mg ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-4-(4-fluorophenyl)-2(R)-hydroxybutyl]-L-prolinetert.butyl ester as a white solid of melting point 163°-164° C.

TheN-[3(S)-(benzyloxyformamido)-4-(4-fluorophenyl)-2(R)-hydroxybutyl]-L-prolinetert.butyl ester used as the starting material was prepared as follows:

(i) In a manner analogous to that described in Example 19(i), 7.70 g ofN-(benzyloxycarbonyl)-3-(4-fluorophenyl)-DL-alanine were treated withisobutyl chloroformate followed by reaction with diazomethane to give7.15 g of benzyl [2-(4-fluorophenyl)-1(RS)-(2-diazoacetyl)ethyl]carbamate as a yellow solid of melting point 97° C.

(ii) Treatment of the above product with hydrogen chloride in a manneranalogous to that described in Example 19(ii) gave 6.70 g of[N-(benzyloxycarbonyl)-3-(4-fluorophenyl)-DL-alanyl]methyl chloridewhich was used without further purification.

(iii) Reduction of 6.30 g of the above product with sodium borohydridein a manner analogous to that described in Example 19(iii) gave 5.83 gof a mixture of alcohols. This mixture was chromatographed on a columnof silica gel using 2% methanol in dichloromethane for the elution togive 0.95 g ofthreo-3-(benzyloxyformamido)-1-chloro-4-(4-fluorophenyl)-2-butanol as awhite solid. Further elution of the column with the same solvent systemgave 3.25 g oferythro-3-(benzyloxyformamido)-1-chloro-4-(4-fluorophenyl)-2-butanol asa white solid of melting point 148°-149° C.

(iv) Treatment of 3.20 g oferythro-3-(benzyloxyformamido)-1-chloro-4-(4-fluorophenyl)-2-butanolwith ethanolic potassium hydroxide solution in a manner analogous tothat described in Example 19(iv) gave 2.03 g oferythro-3-(benzyloxyformamido)-1,2-epoxy-4-(4-fluorophenyl)butane as awhite solid; MS: m/e 315 [M]⁺.

(v) 0.63 g of the above product was treated with L-proline tert.butylester in a manner analogous to that described in Example 19(v). Thecrude product was chromatographed on a column of silica gel using 5%methanol in dichloromethane for the elution. There was obtained 0.26 gofN-[3(R)-(benzyloxyformamido)-4-(4-fluorophenyl)-2(S)-hydroxybutyl]-L-prolinetert.butyl ester as a colourless oil; NMR (CDCl₃, 300 MHz) δ1.47 (9H,s), 1.75-1.95 (4H,m), 2.05-2.15 (1H,m), 2.25-2.35 (1H,m), 2.50-3.00(4H,m), 3.20 (2H,m), 3.58 (1H,m), 3.85 (1H,m), 4.88 (1H,d), 5.00(2H,dd), 6.94 (2H, t), 7.13-7.37 (7H,m).

Further elution with the same solvent system yielded 0.275 g ofN-[3(S)-(benzyloxyformamido)-4-(4-fluorophenyl)-2(R)-hydroxybutyl]-L-prolinetert.butyl ester as a colourless oil; NMR (CDCl₃, 300 MHz) δ1.42 (9H,s),1.75-1.95 (4H,m), 2.05-2.20 (1H,m), 2.54-2.80 (3H,m), 2.85-2.98 (2H,m),3.03-3.24 (2H,m), 3.44 (1H,m), 3.92 (1H,m), 4.88-5.06 (3H,m), 6.93 (2H,t), 7.10-7.40 (7H,m).

EXAMPLE 37

A solution of 730 mg ofN-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-(4-methoxyphenyl)butyl]-L-prolinetert.butyl ester in 20 ml of ethanol was hydrogenated over 50 mg of 10%palladium-on-carbon for 2 hours. The catalyst was removed by filtrationand the filtrate was evaporated to give an oil which was coupled with340 mg of N-(benzyloxycarbonyl)-L-asparagine in a manner analogous tothat described in Example 1. The crude product was chromatographed on acolumn of silica gel using 10% methanol in dichloromethane for theelution. There were obtained 82 mg ofN-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-(4-methoxyphenyl)butyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 613 [M+H]⁺.

TheN-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-(4-methoxyphenyl)butyl]-L-prolinetert.butyl ester used as the starting material was prepared as follows:

(i) In a manner analogous to that described in Example 19(i), 3.10 g ofN-(benzyloxycarbonyl)-3-(4-methoxyphenyl)-L-alanine were treated withisobutyl chloroformate followed by diazomethane to give 2.83 g of benzyl[2-(4-methoxyphenyl)-1(S)-(2-diazoacetyl)ethyl]carbamate as a paleyellow solid of melting point 88°-90° C.

(ii) 2.80 g of the above product were treated with hydrogen chloride ina manner analogous to that described in Example 19(ii) to give 2.85 g of[N-(benzyloxycarbonyl)-3-(4-methoxyphenyl)-L-alanyl]methyl chloride as awhite solid of melting point 107° C.

(iii) The above product was treated with sodium borohydride in a manneranalogous to that described in Example 19(iii). The crude product wastriturated with 30 ml of 5% methanol in dichloromethane to give 1.15 gof 3(S)-(benzyloxyformamido)-1-chloro-4-(4-methoxyphenyl)-2(S)-butanolas a white solid; MS: m/e 363, 365 [M]⁺.

(iv) The above product was treated with ethanolic potassium hydroxidesolution in a manner analogous to that described in Example 19(iv) togive 0.86 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-(4-methoxyphenyl)butane as awhite solid of melting point 86°-87° C.

(v) 0.51 g of the above product was treated with L-proline tert.butylester in a manner analogous to that described in Example 19(v) to give0.63 g ofN-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-(4-methoxyphenyl)butyl]-L-prolinetert.butyl ester as a colourless oil; MS: m/e 498 [M]⁺.

EXAMPLE 38

A solution of 1.58 g of N₂-[3(S)-(benzyloxyformamido)-4-(4-tert.butoxyphenyl)-2(R)-hydroxybutyl]-N.sup.1-tert.butyl-L-prolinamide in 200 ml of ethanol was hydrogenated over 100mg of 10% palladium-on-carbon for 4 hours. The catalyst was removed byfiltration and the filtrate was evaporated to give an oil which wascoupled with 0.74 g of N-(benzyloxycarbonyl)-L-asparagine in a manneranalogous to that described in Example 1. The crude product waschromatographed on a column of silica gel using 10% methanol indichloromethane for the elution. There was obtained 0.59 g of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-4-(4-tert.butoxyphenyl)-2(R)-hydroxybutyl]-N¹-tert.butyl-L-prolinamide as a white solid; MS: m/e 654 [M+H]⁺.

The N₂-[3(S)-(benzyloxyformamido)-4-(4-tert.butoxyphenyl)-2(R)-hydroxybutyl]-N.sup.1-tert.butyl-L-prolinamide used as the starting material was prepared asfollows:

(i) In a manner analogous to that described in Example 19(i),N-(benzyloxycarbonyl)-3-(4-tert.butoxyphenyl)-L-alanine (prepared from7.45 g of the dicyclohexylammonium salt) was treated with isobutylchloroformate followed by reaction with diazomethane to give 3.58 g ofbenzyl [2-(4-tert.butoxyphenyl)-1(S)-(2-diazoacetyl)ethyl]carbamate as ayellow solid of melting point 80°-82° C.

(ii) Treatment of the foregoing carbamate with hydrogen chloride in amanner analogous to that described in Example 19(ii) gave 3.6 g of[N-(benzyloxycarbonyl)-3-(4-tert.butoxyphenyl)-L-alanyl]methyl chloridewhich was used without further purification.

(iii) Reduction of the foregoing chloride with sodium borohydride in amanner analogous to that described in Example 19(iii) gave a mixture ofalcohols which was triturated with hot n-hexane to give 3.14 g of awhite solid. This solid was recrystallized from a mixture of ethylacetate and n-hexane to give 1.88 g of3(S)-(benzyloxyformamido)-4-(4-tert.butoxyphenyl)-1-chloro-2(S)-butanolas a white solid; NMR (CDCl₃, 250 MHz) δ1.35 (9H, s), 2.85-3.05 (3H,m),3.50-3.70 (2H,m), 3.80-3.90 (1H,m), 3.92-4.04 (1H,m), 4.85 (1H,d),5.00-5.10 (2H,m), 6.93 (2H,d), 7.10 (2H,d), 7.25-7.40 (5H,m).

(iv) 5.52 g of the above product were treated with ethanolic potassiumhydroxide solution in a manner analogous to that described in Example19(iv) to give 4.98 g of3(S)-(benzyloxyformamido)-4-(4-tert.butoxyphenyl)-1,2(S)-epoxybutane asa colourless gum: NMR (CDCl₃, 250 MHz) δ1.36 (9H, s), 2.75-3.05 (5H,m),3.76 (1H,m), 4.73 (1H,d), 5.07 (2H, s), 6.95 (2H,d), 7.10 (2H,d),7.25-7.40 (5H,m).

(v) 2.50 g of the above product were treated with N¹-tert.butyl-L-prolinamide in a manner analogous to that described inExample 19(v) to give 1.58 g of N₂-[3(S)-(benzyloxyformamido)-4-(4-tert.butoxyphenyl)-2(R)-hydroxybutyl]-N.sup.1-tert.butyl-L-prolinamide as a colourless oil; MS: m/e 539 [M]⁺.

EXAMPLE 39

A solution of 200 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-4-(4-tert.butoxyphenyl)-2(R)-hydroxybutyl]-N¹-tert.butyl-L-prolinamide in 5 ml of trifluoroacetic acid was stirred atroom temperature for 20 hours. The solution was poured into excesssaturated sodium hydrogen carbonate solution and the mixture wasextracted with three 30 ml portions of dichloromethane and three 30 mlportions of ethyl acetate. The combined extracts were dried overanhydrous sodium sulphate and evaporated to give 100 mg of a colourlessgum. This was chromatographed on a column of silica gel using 10%methanol in dichloromethane for the elution to give 42 mg of aglass-like solid. This solid was triturated with a mixture of 1 ml ofdiethyl ether and 3 ml of n-hexane to give 28 mg of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-(4-hydroxyphenyl)butyl]-N¹-tert.butyl-L-prolinamide trifluoroacetate as a glass-like solid: MS:m/e 598 [M--CF₃ CO₂ ]⁺.

EXAMPLE 40

In a manner analogous to that described in Example 1, from N²-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-L-prolinamide and N-(benzyloxycarbonyl)-L-aspartic acid4-methyl ester there was obtained N₂-[3(S)-[[N-(benzyloxycarbonyl)-4-O-methyl-L-α-aspartyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a glassy solid of melting point 55°-60° C.;MS: m/e 597 [M+H]⁺.

EXAMPLE 41

In a manner analogous to that described in Example 1, from N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide and N-(benzyloxycarbonyl)-L-methionine therewas obtained N²-[3(S)-[[N-(benzyloxycarbonyl)-L-methionyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid of melting point 132°-133° C.

EXAMPLE 42

N₂-[3(S)-[[N,3-bis(Benzyloxycarbonyl)-L-histidyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide was prepared from N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide and N,3-bis(benzyloxycarbonyl)-L-histidine inan analogous manner to that described in Example 1 and was obtained as acolourless glass; MS: m/e 739 [M+H]⁺.

EXAMPLE 43

A solution of 30 mg of N²-[3(S)-[[N,3-bis(benzyloxycarbonyl)-L-histidyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-L-prolinamidein 6 ml of 2M hydrochloric acid was stirred at room temperatureovernight. The mixture was neutralized by the addition of 2M sodiumhydroxide solution and extracted with two 10 ml portions ofdichloromethane. The combined extracts were dried over anhydrous sodiumsulphate and evaporated. The crude product was triturated with diethylether to give 14 mg of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-histidyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid of melting point 100° C.(decomposition); MS: m/e 604 [M]⁺.

EXAMPLE 44

In a manner analogous to that described in Example 21, from 224 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 70 mg of benzoyl chloride and 65 mg ofdiisopropylethylamine there were obtained, after chromatography onsilica gel using 10% methanol in dichloromethane for the elution, 20 mgofN-[3(S)-[(N-benzoyl-L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid from methanol/diethyl ether; MS: m/e553 [M+H]⁺.

EXAMPLE 45

In a manner analogous to that described in Example 27, from 224 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 93 mg of 1-naphthylacetic acid, 68 mg ofhydroxybenzotriazole, 58 mg of N-ethylmorpholine and 113 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using 10% methanol in dichloromethane for the elution, 106 mgofN-[2(R)-hydroxy-3(S)-[[N-[2-(1-naphthyl)acetyl]-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid from methanol/diethyl ether; MS: m/e617 [M+H]⁺.

EXAMPLE 46

In a manner analogous to that described in Example 27, from 493 mg ofN-[3(S)-[[n-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 190 mg of quinaldic acid, 149 mg ofhydroxybenzotriazole, 127 mg of N-ethylmorpholine and 249 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using 10% methanol in dichloromethane for the elution, 167 mgofN-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-L-prolinetert.butyl ester as a white solid from methanol/diethyl ether; MS: m/e604 [M+H]⁺.

EXAMPLE 47

In a manner analogous to that described in Example 27, from 234 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 96 mg of 4-chlorohydrocinnamic acid, 70 mg ofhydroxybenzotriazole, 60 mg of N-ethylmorpholine and 118 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using System G for the elution, 171 mg ofN-[3(S)-[[N-(4-chlorohydrocinnamoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester as a white solid; MS: m/e 615 [M+H]⁺.

EXAMPLE 48

In a manner analogous to that described in Example 27, from 234 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester, 90 mg of 1-isoquinolinecarboxylic acid, 70 mg ofhydroxybenzotriazole, 60 mg of N-ethylmorpholine and 118 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using 10% methanol in dichloromethane for the elution, 146 mgofN-[2(R)-hydroxy-3(S)-[[N-(1-isoquinolylcarbonyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinetert.butyl ester as a pale cream solid from ethyl acetate/n-hexane; MS:m/e 604 [M+H]⁺.

EXAMPLE 49

In a manner analogous to that described in Example 21, from 209 mg of N₂-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 89 mg of 2-naphthoyl chloride and 61 mg ofdiisopropylethylamine there were obtained 191 mg of N¹ -tert.butyl-N²-[2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]-amino]-4-phenylbutyl]-L-prolinamideas a white solid; MS: m/e 602 [M+H]⁺.

The starting material was prepared by hydrogenating N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide; MS: m/e-582 [M+H]⁺. The latter compound wasobtained in a manner analogous to that described in Example 33, butusing N¹ -tert.butyl-L-prolinamide in paragraph (iii) in place of N¹-phenyl-L-prolinamide.

EXAMPLE 50

A solution of 2.798 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-cyclopenta[b]pyrrole-2-carboxamide(four diastereomers) and 2.72 g of N-(benzyloxycarbonyl)-L-asparaginesuccinimide ester in 50 ml of dimethoxyethane was stirred at 20° C. for2 hours and the solvent was then removed by evaporation. The residue wastaken up in ethyl acetate and the solution was washed with aqueoussodium carbonate solution and sodium chloride solution. The organicsolution was evaporated and the residue was chromatographed on silicagel using system G for the elution. There were obtained 434 mg of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamideas a white solid from ethyl acetate; MS: m/e 622 [M+H]⁺ ; Rf: 0.22.

The1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-cyclopenta[b]pyrrole-2-carboxamideused as the starting material was prepared as follows:

(i) A solution of 2.98 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane and 2.1 g ofN-tert.butyl-octahydro-cyclopenta[b]pyrrole-2-carboxamide in 50 ml ofethanol was heated at reflux for 10 hours and the solvent was thenremoved by evaporation. Chromatography on silica gel using System H forthe elution then gave 4.7 g of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-cyclopenta[b]pyrrole-2-carboxamideas a mixture of four diastereomers: MS: m/e 508 [M+H]⁺.

(ii) A solution of 4.6 g of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-cyclopenta[b]pyrrole-2-carboxamide(four diastereomers) in 90 ml of ethanol was hydrogenated over 10%palladium-on-carbon at 20° C. and under atmospheric pressure for 72hours. The catalyst was removed by filtration and the filtrate wasevaporated to give 2.958 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-cyclopenta[b]pyrrole-2-carboxamide(four diastereomers) as a brownish oil; MS: m/e 374 [M+H]⁺.

EXAMPLE 51

In a manner analogous to that described in Example 27, from 310 mg of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamide,111 mg of quinaldic acid, 86 mg of hydroxybenzotriazole, 74 mg ofN-ethylmorpholine and 132 mg of dicyclohexylcarbodiimide there wereobtained, after chromatography on silica gel usingdichloromethane/methanol (9:1) for the elution, 200 mg ofN-tert.butyl-octahydro-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamideas a white solid: MS: m/e 643 [M+H]⁺.

The1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-(3aS,6aS)-cyclopenta-[b]pyrrole-2(S)-carboxamideused as the starting material was prepared as follows:

A solution of 397 mg of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamidein 20 ml of ethanol was hydrogenated over 10% palladium-on-carbon at 20°C. and under atmospheric pressure for 4 hours, The catalyst was removedby filtration and the filtrate was evaporated to give 312 mg of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-octahydro-(3aS,6aS)-cyclopenta[b]pyrrole-2(S)-carboxamideas a pale yellow gum; m/e 488 [M+H]⁺.

EXAMPLE 52

In a manner analogous to that described in Example from 295 mg of N₂-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 204 mg of 6-(benzyloxycarbonyl)-2-naphthoicacid, 20 mg of hydroxybenzotriazole, 77 mg of N-ethylmorpholine and 151mg of dicyclohexylcarbodiimide there were obtained, after chromatographyon silica gel using System G for the elution, 340 mg of N²-[3(S)-[[N-[6-(benzyloxycarbonyl)-2-naphthoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as an off-white solid from ethylacetate/n-hexane; MS: m/e 736 [M+H]⁺.

EXAMPLE 53

294 mg of N₂-[3(S)-[[N-[6-(benzyloxycarbonyl)-2-naphthoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide in 100 ml of isopropanol was hydrogenated over10% palladium-on-carbon at 20° C. and under atmospheric pressure for 16hours. The catalyst was removed by filtration and the filtrate wasevaporated to give 145 mg of N²-[3(S)-[[N-(6-carboxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid from methanol/ethyl acetate;MS: m/e 646 [M+H]⁺.

EXAMPLE 54

In a manner analogous to that described in Example 27, from 295 mg of N₂-[3(S)-[[n-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 171 mg of 4-(benzyloxycarbonyl)benzoic acid,90 mg of hydroxybenzotriazole, 77 mg of N-ethylmorpholine and 151 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using System G for the elution, 390 mg of N₂-[3(S)-[[N-[4-(benzyloxycarbonyl)benzoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as an off-white solid from ethylacetate/n-hexane; MS: m/e 686 [M+H]⁺.

EXAMPLE 55

274 mg of N²-[3(S)-[[N-[4-(benzyloxycarbonyl)benzoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide in 100 ml of isopropanol were hydrogenatedover 10% palladium-on-carbon at 20° C. and under atmospheric pressurefor 16 hours. The catalyst was removed by filtration and the filtratewas evaporated to give 193 mg of N₂-[3(S)-[[N-(4-carboxybenzoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid from methanol/ethyl acetate;MS: m/e 596 [M+H]⁺.

EXAMPLE 56

In a manner analogous to that described in Example 27, from 228 mg of N₂-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 98 mg of 4-nitrocinnamic acid, 69 mg ofhydroxybenzotriazole, 59 mg of N-ethylmorpholine and 116 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using dichloromethane/methanol (9:1) for the elution, 68 mgof N¹ -tert.butyl-N²-[2(R)-hydroxy-3(S)-[[N-(4-nitrocinnamoyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinamideas a white solid from methanol/diethyl ether; MS: m/e 623 [M+H]⁺.

EXAMPLE 57

A solution of 200 mg of N¹ -tert.butyl-N₂-[2(R)-hydroxy-3(S)-[[N-(4-nitrocinnamoyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinamidein 20 ml of ethanol was hydrogenated over 10% palladium-on-carbon at 20°C. and under atmospheric pressure for 5 hours. The catalyst was removedby filtration and the filtrate was evaporated. The residue waschromatographed on silica gel using dichloromethane/methanol (9:1) forthe elution to give 40 mg of N²-[3(S)-[[N-(4-aminohydrocinnamoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid from methanol/ethyl acetate;MS: m/e 595 [M+H]⁺.

EXAMPLE 58

In a manner analogous to that described in Example 27, from 418 mg of N²-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 205 mg of1-acetyl-1,2,3,4-tetrahydro-2(R,S)-quinoline-carboxylic acid, 127 mg ofhydroxybenzotriazole, 108 mg of N-ethylmorpholine and 212 mg ofdicyclohexylcarbodiimide there were obtained, after purification bychromatography on silica gel using dichloromethane/methanol (9:1) forthe elution, 220 mg of N²-[3(S)-[[N-[(1-acetyl-1,2,3,4-tetrahydro-2(RS)-quinolyl)carbonyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl-N¹-tert.butyl-L-prolinamide (two diastereomers) as a white solid fromdichloromethane/diethyl ether; MS: m/e 649 [M+H]⁺.

EXAMPLE 59

In a manner analogous to that described in Example 27, from 418 mg of N²-[3(S)-[(L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 179 mg of1,2,3,4-tetrahydro-1-oxo-3(RS)-isoquinolinecarboxylic acid, 127 mg ofhydroxybenzotriazole, 108 mg of N-ethylmorpholine and 212 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using System I for the elution, 132 mg of N¹ -tert.butyl-N²-[3(S)-[[N-[(1,2,3,4-tetrahydro-1-oxo-3(R orS)-isoquinolyl)carbonyl]-L-asparaginyl]amino]-2(R)-hydroxyphenylbutyl]-L-prolinamide(diastereomer A) as a white solid from methanol/diethyl ether, MS: m/e621 [M+H]⁺, and 66 mg of N¹ -tert.butyl-N₂-[3(S)-[[N-[(1,2,3,4-tetrahydro-1-oxo-3(R orS)-isoquinolyl)carbonyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinamide(diastereomer B) as a white solid from methanol/diethyl ether; MS: m/e621 [M+H]⁺.

EXAMPLE 60

In a manner analogous to that described in Example 27, from 418 mg of N₂-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 218 mg of2-acetamido-1,2,3,4-tetrahydro-2-naphthoic acid, 127 mg ofhydroxybenzotriazole, 108 mg of N-ethylmorpholine and 212 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using dichloromethane/methanol (9:1) for the elution, 142 mgof N₂ -[3(S)-[[N-[2(R orS)-acetamido-1,2,3,4-tetrahydro-2-naphthoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide (diastereomer A) as a white solid, MS: m/e 663[M+H]⁺, and 80 mg of N² -[3(S)-[[N-[2(R orS)-acetamido-1,2,3,4-tetrahydro-2-naphthoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide (diastereomer B) as a white solid; MS: m/e 663[M+H]⁺.

EXAMPLE 61

In a manner analogous to that described in Example 27, from 209 mg of N²-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 81 mg of quinaldicacid, 63 mg ofhydroxybenzotriazole, 54 mg of N-ethylmorpholine and 106 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using dichloromethane/methanol (9:1) for the elution, 88 mgof N₂-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-N¹-tert.butyl-L-prolinamide as a white solid; MS: m/e 603 [M+H]⁺.

EXAMPLE 62

A solution of 844 mg of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideand 882 mg of N-(benzyloxycarbonyl)-L-asparagine succinimide ester in 25ml of tetrahydrofuran was stirred at 20° C. for 16 hours and thenevaporated. The residue was dissolved in 50 ml of dichloromethane andthe organic solution was washed with saturated sodium chloride solutionand water. The solvent was removed by evaporation and the residue waschromatographed on silica gel using dichloromethane/methanol (9:1) forthe elution. There were obtained 461 mg of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideas a pale cream solid; MS: m/e 596 [M+H]⁺.

The1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideused as the starting material was prepared as follows:

(i) 2.576 g of N-tert.butyl-2(S)-piperidinecarboxamide and 4.158 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in 70 ml ofethanol was heated at reflux for 16 hours. The solvent was removed byevaporation and the residue was dissolved in 100 ml of diethyl ether andtreated with 10 g of activated magnesium silicate. The solvent was thenremoved by evaporation and there were obtained 6.16 g of1-[3(S)l(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideas a colourless glass; MS: m/e 482 [M+H]⁺.

(ii) A solution of 1.25 g of1-3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamidein 80 ml of ethanol was hydrogenated over 10% palladium-on-carbon at 20°C. and under atmospheric pressure for 16 hours. The catalyst was removedby filtration and the filtrate was evaporated to give 0.844 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideas a pale yellow gum; MS: m/e 348 [M+H]⁺.

EXAMPLE 63

In a manner analogous to that described in Example 21, from 226 mg of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,93 mg of 2-naphthoyl chloride and 63 mg of diisopropylethylamine therewere obtained, after chromatography on silica gel usingdichloromethane/methanol (9:1) for the elution, 76 mg ofN-tert.butyl-1-[2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-2(S)-piperidinecarboxamideas a white solid; MS: m/e 616 [M+H]⁺.

The1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideused as the starting material was prepared by hydrogenating1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide.

EXAMPLE 64

In a manner, analogous to that described in Example 27, from 226 mg of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,85 mg of quinaldic acid, 56 mg of N-ethylmorpholine, 66 mg ofhydroxybenzotriazole and 111 mg of dicyclohexylcarbodiimide there wereobtained, after chromatography on silica gel usingdichloromethane/methanol (9:1) for the elution, 100 mg of N-tert.butyl1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2(S)-piperidinecarboxamideas a white solid; MS: m/e 617 [M+H]⁺.

EXAMPLE 65

In a manner analogous to that described in Example 27, from 228 mg of N²-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 88 mg of 3-isoquinolinecarboxylic acid, 69 mgof hydroxybenzotriazole, 59 mg of N-ethylmorpholine and 116 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using dichloromethane/methanol (9:1) for the elution, 88 mgof N¹ -tert.butyl-N₂-[2(R)-hydroxy-3(S)-[[N-(3-isoquinolylcarbonyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinamideas a white solid; MS: m/e 603 [M+H]⁺.

EXAMPLE 66

In a manner analogous to that described in Example 27, from 228 mg of N₂-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 88 mg of 3-quinolinecarboxylic acid, 69 mg ofhydroxybenzotriazole, 59 mg of N-ethylmorpholine and 116 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using dichloromethane/methanol (9:1) for the elution, 86 mgof N¹ -tert.butyl-N²-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(3-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-L-prolinamideas a white solid; MS: m/e 603 [M+H]⁺.

EXAMPLE 67

In a manner analogous to that described in Example 27, from 295 mg of N²-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide, 214 mg of 3-(benzyloxyformamido)-2-naphthoicacid, 90 mg of hydroxybenzotriazole, 77 mg of N-ethylmorpholine and 151mg of dicyclohexylcarbodiimide there were obtained, after chromatographyon silica gel using System G for the elution, 380 mg of N²-[3(S)-[[N-[3-(benzyloxyformamido)-2-naphthoyl]-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid from methanol; MS: m/e 751[M+H]⁺.

EXAMPLE 68

A solution of 216 mg of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide (diastereomer A), 151 mg ofN-(benzyloxycarbonyl)-L-asparagine, 76 mg of hydroxybenzotriazole, 65 mgof N-ethylmorpholine and 128 mg of dicyclohexylcarbodiimide in 10 ml oftetrahydrofuran was stirred at 20° C. for 16 hours. The solvent wasremoved by evaporation and the residue was chromatographed on silica gelusing dichloromethane/methanol (92:8) for the elution. There wereobtained 70 mg of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(R or S)-carboxamide (diastereomer A) as a pale cream solid; MS: m/e 630[M+H]⁺.

The1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide used as the starting material was prepared as follows:

(i) A solution of 891 mg of N-tert.butyl-2,3-dihydro-1H-indole-2(R orS)-carboxamide and 654 mg of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in 5 ml ofdimethylformamide was heated to 140° C. for 40 hours. The solvent wasremoved by evaporation and the residue was chromatographed on silica gelusing diethyl ether/n-hexane (2:1) for the elution. There were obtained300 mg of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide (diastereomer A) as a pale yellow oil, m/e 516 [M+H]⁺,and 290 mg of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide (diastereomer B) as a pale yellow oil; MS: m/e 516[M+H]⁺.

(ii) A solution of 300 mg of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide (diastereomer A) in 20 ml of ethanol was hydrogenatedover 10% palladium-on-carbon at 20° C. and under atmospheric pressurefor 16 hours. The catalyst was removed by filtration and the filtratewas evaporated to give 216 mg of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide as a brownish oil.

EXAMPLE 69

In a manner analogous to that described in Example 68, from 223 mg of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide (diastereomer B), 151 mg ofN-(benzyloxycarbonyl)-L-asparagine, 76 mg of hydroxybenzotriazole, 65 mgof N-ethylmorpholine and 128 mg of dicyclohexylcarbodiimide there wereobtained 210 mg of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2,3-dihydro-1H-indole-2(Ror S)-carboxamide (diastereomer B) as a pale cream solid; MS: m/e 630[M+H]⁺.

EXAMPLE 70

A solution of 0.422 g of N₂-[2(R)-hydroxy,4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-N¹-tert.butyl-L-prolinamide and 142 mg of 3-chloroperbenzoic acid in 10 mlof dichloromethane was stirred at 20° C. for 1 hour. The solvent wasthen removed by evaporation and the residue was chromatographed onsilica gel using dichloromethane/methanol (9:1) for the elution to give289 mg of N¹ -tert.butyl-N₂-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-L-prolinamideN₂ -oxide as a white solid; MS: m/e 619 [M+H]⁺.

EXAMPLE 71

A solution of 0.123 mg of N¹ -tert.butyl-N₂-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-L-prolinamideN₂ -oxide and 35 mg of 3-chloroperbenzoic acid in 5 ml ofdichloromethane was stirred at 25° C. for 24 hours. A further 70 mg of3-chloroperbenzoic acid was added and the mixture was stirred at 20° C.for 48 hours. The solution was washed with aqueous sodium carbonatesolution and sodium chloride solution and the solvent was then removedby evaporation. The residue was chromatographed on silica gel usingSystem J for the elution to give 70 mg of N¹ -tert.butyl-N²-[2(R)-hydroxy-3(S)-[[N-(1-oxido-2-quinolylcarbonyl)-L-asparaginyl]amino]-4-phenylbutyl]-L-prolinamideN² -oxide as an off-white solid from ethyl acetate; MS: m/e 635 [M+H]⁺.

EXAMPLE 72

A solution of 98 mg of3-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamidedihydrobromide in 0.5 ml of dry dimethylformamide was stirred and cooledto -10° C. in an ice/salt bath while there were added 44 mg ofN-ethylmorpholine followed by 76 mg ofN-(benzyloxycarbonyl)-L-asparagine succinimide ester. The mixture wasallowed to warm to room temperature and was then stirred overnight.Dimethylformamide was removed by evaporation under reduced pressure andthe residue was dissolved in chloroform. The solution was washed withwater, then dried over sodium sulphate and evaporated. The residual gumwas purified by flash chromatography on silica gel using 4% methanol indichloromethane for the elution. There were obtained 66 mg of3-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamideas a colourless foam. Analytically pure product was obtained byrecrystallization from ethyl acetate; MS: m/e 600 [M+H]⁺.

The3-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamidedihydrobromide used as the starting material was prepared as follows:

(i) A solution of 2.67 g ofN-benzyloxycarbonyl-4(R)-thiazolidinecarboxylic acid in 42 ml of drytetrahydrofuran was stirred and cooled to -15° C. in an ice/salt bathwhile there were added 1.15 g of N-ethylmorpholine followed after 2minutes by 1.87 g of isobutyl chloroformate. After a further 3 minutes0.73 g of tert.butylamine was added dropwise and the mixture was thenallowed to warm to room temperature and was stirred overnight.Tetrahydrofuran was removed by evaporation under reduced pressure andthe residue was dissolved in dichloromethane. The solution was washed insequence with water, 10% citric acid solution, water, saturated sodiumbicarbonate solution and water, then dried over anhydrous sodiumsulphate, filtered and evaporated to give 2.85 g of3-(benzyloxycarbonyl)-N-tert.butyl-4(R)-thiazolidinecarboxamide as asolid which melted at 96°-98° C. after recrystallization from diethylether/n-hexane.

(ii) A mixture of 3.2 g of3-(benzyloxycarbonyl)-N-tert.butyl-4(R)-thiazolidinecarboxamide and 10ml of 32% (w/w) hydrogen bromide in glacial acetic acid was stirred atroom temperature for 2 hours. The resulting solution was poured intodiethyl ether and the precipitated hydrobromide salt of the product wasfiltered off, washed with diethyl ether and then dissolved in water. Thesolution was made alkaline by the addition of 1M sodium hydroxidesolution and extracted twice with dichloromethane. The combineddichloromethane extracts were washed with water, dried over anhydroussodium sulphate, filtered and evaporated to give 1.57 g ofN-tert.butyl-4(R)-thiazolidinecarboxamide as a crystalline solid ofmelting point 68°-71° C.

(iii) A solution of 3.2 g of N-tert.butyl-4(R)-thiazolidinecarboxamideand 5.0 g of 3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in130 ml of isopropanol was stirred under argon and heated at 90° C. underreflux for 3 days. The solution was evaporated under reduced pressureand the residue was purified by flash chromatography on silica gel usingdiethyl ethyl/n-hexane (2:1) for the elution. There were obtained 3.47 gof3-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamide.Recrystallization from ethyl acetate gave analytically pure material ofmelting point 62°-65° C.

(iv) A mixture of 2 g of3-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamideand 4.1 ml of 32% (w/w) hydrogen bromide in glacial acetic acid wasstirred at room temperature for 1 hour. The resulting solution wasdiluted with anhydrous diethyl ether and the precipitated, product wasrapidly filtered off and washed with fresh diethyl ether. There wereobtained 2.01 g of3-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamidedihydrobromide as a white solid; ¹ H NMR (300 MHz): δ(D₂ O) 1.37 (9H,s), 3.0 (2H, m), 3.09 (2H, dq), 3.29 (2H, d), 3.85 (2H, m), 4.16 (1H,m), 4.3 (2H, q) and 7.42 (5H, m) ppm.

EXAMPLE 73

In a manner analogous to that described in Example 21, from 110 mg of3-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamide,46 mg of 2-naphthoyl chloride and 31 mg of diisopropylethylamine therewere obtained, after flash chromatography on silica gel using 2%methanol in dichloromethane for the elution, 83 mg ofN-tert.butyl-3-[2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-4(R)-thiazolidinecarboxamide;MS: m/e 620 [M+H]⁺.

The3-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamideused as the starting material was prepared as follows:

A mixture of 170 mg of3-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamideand 0.3 ml of 32% (w/w) hydrogen bromide in glacial acetic acid wasstirred at room temperature for 1 hour. The solution was diluted withdiethyl ether and the precipitated solid was rapidly filtered off,washed with diethyl ether and dissolved in water. The solution was madealkaline by the addition of potassium carbonate and extracted with threeportions of chloroform. The combined chloroform extracts were dried overanhydrous sodium sulphate, filtered and evaporated to give 110 mg of3-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-4(R)-thiazolidinecarboxamide;MS: m/e 466 [M+H]⁺.

EXAMPLE 74

A solution of 330 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-4-(4-tert.butoxyphenyl)-2(R)-hydroxybutyl]-N¹-tert.butyl-L-prolinamide in 30 ml of ethanol was hydrogenated over 50mg of 10% palladium-on-carbon catalyst for 3 hours. The catalyst wasremoved by filtration and the filtrate was evaporated. The residue wasdissolved in dichloromethane and treated with 72 mg ofdiisopropylethylamine and 106 mg of 2-naphthoyl chloride in a manneranalogous to that described in Example 21 to give 165 mg of N₂-[4-(4-tert.butoxyphenyl)-2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]butyl]-N¹-tert.butyl-L-prolinamide as an off-white solid.

EXAMPLE 75

A solution of 90 mg of N₂-[4-(4-tert.butoxyphenyl)-2(R)-hydroxy-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]butyl]-N¹-tert.butyl-L-prolinamide in 40 ml of 3.5M hydrogen chloride in ethylacetate was stirred at room temperature for 30 minutes. The solvent wasremoved by evaporation and the residue was triturated with diethyl etherand filtered to give 80 mg of N₂-[2(R)-hydroxy-4-(4-hydroxyphenyl)-3(S)-[[N-(2-naphthoyl)-L-asparaginyl]amino]butyl]-N-tert.butyl-L-prolinamide hydrochloride as a white solid of meltingpoint 171°-174° C.

EXAMPLE 76

A solution of 59 mg of2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]-indole-1-carboxamide(isomer A) and 71 mg of N-(benzyloxycarbonyl)-L-asparagine succinimideester in 3 ml of dry tetrahydrofuran was stirred at room temperature for16 hours and then evaporated. The residue was dissolved in ofdichloromethane and the solution was washed twice with water and twicewith saturated aqueous sodium bicarbonate solution. The solvent wasremoved by evaporation under reduced pressure to give 103 mg of crudeproduct. 32 mg of this product were purified by reverse phase highpressure liquid chromatography using 55% 0.05M ammonium formate inacetonitrile for the elution. There were obtained 9.9 mg of2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1(Ror S)-carboxamide (isomer A) as a colourless oil; MS: m/e 683 [M+H]⁺.

2-[3(S)-[[N-(Benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide(isomer B) was prepared from2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1(Ror S)-carboxamide (isomer B) in a manner analogous to isomer A above andwas purified in the same manner to give a colourless oil; MS: m/e 683[M+H]⁺.

The2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamidesused as the starting materials were prepared as follows:

(i) To a solution, cooled in ice, of 3.0 g of1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxylic acid in 7 ml of 2Msodium hydroxide solution, 17 ml of water and 17 ml of dioxan were addedin alternating portions 12.5 ml of 2M sodium hydroxide solution and asolution of 3.6 ml of benzyl chloroformate in 9 ml of dioxan. Aftercompletion of the additions the mixture was stirred at room temperatureovernight and the dioxan-was then removed by evaporation under reducedpressure. The solution obtained was diluted with water, washed twicewith diethyl ether, acidified with 25 ml of 1M sulphuric acid andextracted three times with ethyl acetate. The organic extracts werecombined, washed twice with water, dried over anhydrous magnesiumsulphate and evaporated to give 4.54 g of2-benzyloxycarbonyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxylicacid, MS: m/e 351 [M+H]⁺, which was used without purification.

(ii) A solution of 4.54 g of2-benzyloxycarbonyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxylicacid in 45 ml of anhydrous tetrahydrofuran was cooled, while protectingwith a drying tube, in an ice/acetone bath. 1.82 ml of N-ethylmorpholineand 1.82 ml of isobutyl chloroformate were added, the mixture wasstirred for 10 minutes and then 2.1 ml of tert.butylamine were added.The mixture was then stirred at 0° C. for 40 minutes and at roomtemperature for 45 minutes, diluted with ethyl acetate and washed twicewith water, twice with 0.5M sodium hydroxide solution and again withwater. The solution was dried over magnesium sulphate and evaporated todryness. Recrystallization from ethyl acetate and n-hexane gave 1.21 gof tert.butyl2-benzyloxycarbonyl-1,2,3,4-tetrahydropyrido-[3,4-b]indole-1-carboxamide;MS: m/e 406 [M+H]⁺. A further 0.76 g of identical material was obtainedby subjecting the mother liquors to chromatography on silica gel usingethyl acetate/n-hexane (1:1) for the elution.

(iii) 1.62 g oftert.butyl-benzyloxycarbonyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamidewere dissolved in 10 ml of a 45% solution of hydrogen bromide in aceticacid. After 30 minutes the solution was evaporated and the residue wastaken up in water. After filtration the filtrate was washed three timeswith diethyl ether and then neutralized by the addition of saturatedaqueous sodium bicarbonate solution. The product was extracted from thenow turbid aqueous layer with ethyl acetate. The combined organicextracts were dried over anhydrous magnesium sulphate and evaporated togive 0.84 g of tert.butyl1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide as a whitecrystalline solid of melting point 144° C.

(iv) A solution of 0.60 g of tert.butyl1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide and 0.66 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in 20 ml ofmethanol was heated at reflux under argon for 16 hours and thenevaporated to give a clear oil. The two diastereomeric products wereseparated by flash chromatography on silica gel using n-hexane/ethylacetate (3:1) for the elution. There were obtained 268 mg of isomer A of2-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide,MS: m/e 569 [M+H]⁺, and 65 mg of isomer B of the same compound; MS: m/e569 [M+H]⁺.

(v) A solution of 150 mg of2-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide(isomer A) in 5 ml of ethanol was hydrogenated under a pressure of 3.4atmospheres over 10% palladium-on-charcoal at 20° C. for 16 hours. Thecatalyst was removed by filtration and the filtrate was evaporated togive 120 mg of2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide(isomer A); MS: m/e 435 [M+H]⁺.

2-[3(S)-Amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide(isomer B) MS: m/e 435 [M+H]⁺, was prepared in an analogous manner from2-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1-carboxamide(isomer B).

EXAMPLE 77

In a manner analogous to that described in Example 70, from 154 mg ofN-tert.butyl-1-[2(R)-hydroxy-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino-4-phenylbutyl]-2(S)-piperidinecarboxamideand 51 mg of 3-chloroperbenzoic acid there were obtained 114 mg ofN-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2-piperidinecarboxamide1-oxide as a white solid; MS: m/e 633 [M+H]⁺.

EXAMPLE 78

In a manner analogous to that described in Example 27, from 230 mg ofI-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,139 mg of 3-benzyloxy-2-naphthoic acid, 113 mg ofdicyclohexylcarbodiimide, 68 mg of hydroxybenzotriazole and 58 mg ofN-ethylmorpholine there were obtained, after chromatography on silicagel using System G for the elution, 242 mg of1-[3(S)-[[N-(3-benzyloxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideas a foam; MS: m/e 722 [M+H]⁺.

EXAMPLE 79

A solution of 181 mg of1-[3(S)-[[N-(3-benzyloxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamidein 5 ml of ethanol was hydrogenated over 10% palladium-on-carbon at 20°C. and under atmospheric pressure for 16 hours. The catalyst was removedby filtration and the filtrate was evaporated. After trituration withdiethyl ether there were obtained 110 mg ofN-tert.butyl-1-[3(S)-[[N-(3-hydroxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-2(S)-piperidinecarboxamideas a pale yellow solid; MS: m/e 632 [M+H]⁺.

EXAMPLE 80

In a manner analogous to that described in Example 27, from 400 mg ofcis-1-[3(8)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide, 266 mg ofN-(benzyloxycarbonyl)-L-asparagine, 226 mg of dicyclohexylcarbodiimide,135 mg of hydroxybenzotriazole and 115 mg of N-ethylmorpholine therewere obtained, after chromatography on silica gel usingdichloromethane/methanol (94:6) for the elution, 225 mg ofcis-1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide as a white solid; MS: m/e 650 [M+H]⁺.

Thecis-1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide used as the starting material was prepared asfollows:

(i) A solution of 2.376 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane and 1.904 g ofcis-N-tert.butyl-decahydro-2(R,S)-quinolinecarboxamide in 32 ml ofethanol was stirred at 80° C. for 24 hours. A further 0.474 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane was added in twoportions and the mixture was stirred at 80° C. for a further 5 hours.The solvent was removed by evaporation and the residue waschromatographed on silica gel using dichloromethane/methanol (97.5:2.5)for the elution to give 1.17 g ofcis-1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide (isomer A) as a white solid from diethylether/n-hexane, MS: m/e 536 [M+H]⁺, and 1.146 g ofcis-1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide (isomer B) as a pale yellow gum; MS: m/e 536[M+H]⁺.

(ii) A solution of 0.535 g ofcis-1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide (isomer B) in 25 ml of ethanol washydrogenated over 10% palladium-on-carbon at 20° C. and underatmospheric pressure for 16 hours. The catalyst was removed byfiltration and the filtrate was evaporated to give 400 mg ofcis-1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide as a colourless gum.

EXAMPLE 81

A solution of 561 mg oftrans-2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamideand 372 mg of N-(benzyloxycarbonyl)-L-asparagine in 20 ml of drytetrahydrofuran is cooled in an ice/salt mixture. 189 mg ofhydroxybenzotriazole, 161 mg of N-ethylmorpholine and 317 mg ofdicyclohexylcarbodiimide are added and the mixture is stirred for 16hours. The mixture is then diluted with ethyl acetate and filtered. Thefiltrate is washed with aqueous sodium bicarbonate solution and sodiumchloride solution. The solvent is removed by evaporation and the residueis chromatographed on silica gel using dichloromethane/methanol (9:1)for the elution to givetrans-2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamide

Thetrans-2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamideused as the starting material is prepared as follows:

(i) A solution of 440 mg oftrans-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamide and549 mg of 3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in 6 mlof ethanol is stirred at 60° C. for 7 hours. A further 54 mg of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane are added and thesolution is stirred at 20° C. for 16 hours. The solvent is removed byevaporation and the residue is chromatographed on silica gel usingsystem H for the elution to givetrans-2-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl-N-tert.butyldecahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamide.

(ii) A solution of 747 mg oftrans-2-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamidein 40 ml of ethanol is hydrogenated over 10% palladium-on-carbon at 20°C. and under atmospheric pressure for 5 hours. The catalyst is removedby filtration and the filtrate is evaporated to givetrans-2-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamideas a buff coloured solid.

EXAMPLE 82

In a manner analogous to that described in Example 27, from 276 mg of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,167 mg of 1-benzyloxy-2-naphthoic acid, 81 mg of hydroxybenzotriazole,69 mg of N-ethylmorpholine and 136 mg of dicyclohexylcarbodiimide therewere obtained, after chromatography on silica gel usingdichloromethane/methanol (9:1) for the elution, 97 mg of1-[3(S)-[[N-(1-benzyloxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideas a white solid from methanol/diethyl ether: MS: m/e 722 [M+H]⁺.

EXAMPLE 83

In a manner analogous to that described in Example 79, from 119 mg of1-[3(S)-[[N-(1-benzyloxy-2-naphthoyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamidethere were obtained, after chromatography on activated magnesiumsilicate using dichloromethane/methanol (9:1) for the elution, 67 mg ofN-tert.butyl-1-[2(R)-hydroxy-3(S)-[[N-(1-hydroxy-2-naphthoyl)-L-asparaginyl]amino]-4-phenylbutyl]-2(S)-piperidinecarboxamideas a white solid; MS: m/e 631 [M+H]⁺.

EXAMPLE 84

In a manner analogous to that described in Example 27, from 276 mg of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamide,183 mg of 3-(benzyloxycarbonyl)-2-naphthoic acid, 81 mg ofhydroxybenzotriazole, 138 mg of N-ethylmorpholine and 136 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using System J for the elution, 98 mg ofN-tert.butyl-1-[3(S)-[1(S)-(2,3-dihydro-1,3-dioxo-1H-benz[f]isoindol-2-yl)-3-carbamoylpropionamido]-2(R)-hydroxy-4-phenylbutyl]-2(S)-piperidinecarboxamide;MS: m/e 642 [M+H]⁺.

EXAMPLE 85

A solution of 650 mg of N² -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide and 538 mg ofN-(benzyloxycarbonyl)-S-methyl-L-cysteine in 20 ml of drytetrahydrofuran was cooled in an ice/salt mixture. 270 mg ofhydroxybenzotriazole, 230 mg of N-ethylmorpholine and 412 mg ofdicyclohexylcarbodiimide were added and the mixture was stirred for 16hours. The mixture was diluted with ethyl acetate and filtered. Thefiltrate was washed with aqueous sodium bicarbonate solution and sodiumchloride solution. The solvent was removed by evaporation and theresidue was chromatographed on silica gel using System G for the elutionto give 800 mg of N₂-[3(S)-[N-(benzyloxycarbonyl)-S-methyl-L-cysteinyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide; MS: m/e 585 [M+H]⁺.

The N² -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide used as the starting material was prepared inan analogous manner to the starting material of Example 33, but using N¹-tert.butyl-L-prolinamide in paragraph (iii) in place of N¹-phenyl-L-prolinamide.

EXAMPLE 86

A solution of 193 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-S-methyl-L-cysteinyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide in 2 ml of methanol was cooled to -70° C. Asolution of 62 mg of 3-chloroperbenzoic acid in 5 ml of methanol wasadded and the mixture was stirred at -70° C. for 30 minutes. The solventwas then removed by evaporation and the residue was chromatographed onsilica gel using System G for the elution to give 62 mg of N₂-[3(S)-[[N-(benzyloxycarbonyl)-3-(methylsulphinyl)-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a 1:1 mixture of diastereomers; MS: m/e 601[M+H]⁺.

EXAMPLE 87

A solution of 450 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-S-methyl-L-cysteinyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide in 4 ml of methanol was cooled to -70° C. 166mg of 3-chloroperbenzoic acid were added portionwise over a period of 10minutes, the solution was stirred at -70° C. for 15 minutes and thenallowed to warm to 20° C. The solution was again cooled to -70° C., afurther 33 mg of 3-chloroperbenzoic acid were added and the mixture wasstirred at -70° C. for 30 minutes. The solvent was removed byevaporation and the residue was partitioned between dichloromethane and2N sodium hydroxide solution. The organic phase was evaporated and theresidue was chromatographed on silica gel using System G for the elutionto give 100 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-3-(methylsulphinyl)-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-L-prolinamide N₂ -oxide (diastereomer A), MS: m/e 617[M+H]⁺, and 154 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-3-(methylsulphinyl)-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide N₂ -oxide (diastereomer B); MS: m/e 617[M+H]⁺.

EXAMPLE 88

A solution of 154 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-3-(methylsulphinyl)-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide N² -oxide (two diastereomers) in 5 ml ofmethanol was treated with 84 mg of 3-chloroperbenzoic acid and thesolution was stirred at 20° C. for 16 hours. The solvent was removed byevaporation and the residue was partitioned between dichloromethane and2M sodium hydroxide solution. The organic phase was evaporated and theresidue was crystallized from ethyl acetate/n-hexane to give 28 mg of N²-[3(S)-[[N-(benzyloxycarbonyl)-3-(methylsulphonyl)-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide N₂ -oxide monohydrate; MS: m/e 633 [M+H]⁺.

EXAMPLE 89

In a manner analogous to that described in Example 27, from 400 mg ofcis-1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide, 266 mg ofN-(benzyloxycarbonyl)-L-asparagine, 226 mg of dicyclohexylcarbodiimide,135 mg of hydroxybenzotriazole and 115 mg of N-ethylmorpholine therewere obtained, after chromatography on silica gel usingdichloromethane/methanol (94:6) for the elution, 225 mg ofcis-1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide as a white solid; MS: m/e 650 [M+H]⁺.

Thecis-1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide used as the starting material was prepared asfollows:

(i) A solution of 2.376 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane and 1.904 g ofcis-N-tert.butyl-decahydro-2(R,S)-quinolinecarboxamide in 32 ml ofethanol was stirred at 80° C. for 24 hours. A further 0.474 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane was added in twoportions and the mixture was stirred at 80° C. for a further 5 hours.The solvent was removed by evaporation and the residue waschromatographed on silica gel using dichloromethane/methanol (97.5:2.5)for the elution to give 1.17 g ofcis-1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-guinolinecarboxamide (isomer A) as a white solid from diethylether/n-hexane, MS: m/e 536 [M+H]⁺, and 1.146 g ofcis-1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide (isomer B) as a pale yellow gum; MS: m/e 536[M+H]⁺.

(ii) A solution of 0.535 g ofcis-1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide (isomer B) in 25 ml of ethanol washydrogenated over 10% palladium-on-carbon at 20° C. and underatmospheric pressure for 16 hours. The catalyst was removed byfiltration and the filtrate was evaporated to give 400 mg ofcis-1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-2(Ror S)-quinolinecarboxamide as a colourless gum.

EXAMPLE 90

In a manner analogous to that described in Example 27, from 27 mg ofN-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester and 11.3 mg of 2-indolecarboxylic acid there wereobtained 15 mg ofN-[3(S)-[[N-(2-indolylcarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-L-prolinetert.butyl ester; MS: m/e 592 [M+H]⁺.

EXAMPLE 91

In a manner analogous to that described in Example 72, from 240 mg of3-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-tetrahydro-2H-1,3-thiazine-4(Rand S)-carboxamide and 234 mg of N-(benzyloxycarbonyl)-L-asparaginesuccinimide ester there were obtained 162 mg of3-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-tetrahydro-2H-1,3-thiazine-4(Rand S)-carboxamide as a mixture of two diastereomers. Flashchromatography on silica gel using 3% methanol in dichloromethane forthe elution gave 20 mg of the less polar diastereomer (isomer A), MS:m/e 614 [M+H]⁺, and using 5% methanol in dichloromethane gave 32 mg ofthe more polar diastereomer (isomer B): MS: m/e 614 [M+H]⁺.

The3-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-tetrahydro-2H-1,3-thiazine-4(Rand S)-carboxamide used as the starting material was prepared from theknown tetrahydro-2H-1,3-thiazine-4(R and S)-carboxylic acid byN-benzyloxycarbonylation in a known manner, subsequent reaction in amanner analogous to that described in Example 72(i)-(iv) andbasification of the dihydrobromide obtained with sodium bicarbonatesolution.

EXAMPLE 92

0.122 g of N-(benzyloxycarbonyl)-3-cyano-L-alanine was dissolved in 2 mlof dry dimethyformamide. The solution was stirred and cooled in anice/salt bath and treated with 0.066 g of hydroxybenzotriazole and 0.1 gof dicyclohexylcarbodiimide. The mixture was stirred for 5 minutes andthen treated with 0.163 g of N₂-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -tert.butyl-L-prolinamide in2.5 ml of dry dichloromethane. The mixture was allowed to warm to roomtemperature and was then stirred overnight. The resultingdicyclohexylurea was filtered off and washed with methylene chloride.The combined filtrate and washings were evaporated at 40° C. in a vacuumto give an oil which was partitioned between ethyl acetate and water.The organic phase was washed in sequence with saturated aqueous sodiumbicarbonate solution and saturated sodium chloride solution and thendried over sodium sulphate. The solvent was removed by evaporation togive an oil which was chromatographed on silica gel using 14% methanolin dichloromethane for the elution. There were thus obtained 50 mg ofproduct which was recrystallized from diethyl ether/n-hexane to give0.045 g of N²-[3(S)-[[N-(benzyloxycarbonyl)-3-cyano-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide hemihydrate as a solid of melting point65°-70° C. (softening).

The N² -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide used as the starting material was prepared asfollows:

(i) A solution of 0.425 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane and 0.244 g ofL-proline tert.butylamide in 10 ml of dry isopropanol was heated at 80°C. for 20 hours. The solvent was removed by evaporation in a vacuum andthe residue was chromatographed on silica gel using 5% methanol indichloromethane for the elution. There was obtained 0.44 g of N²-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white foam.

(ii) A solution of 0.46 g of N²-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide in 40 ml of ethanol was hydrogenated over 40mg of 10% palladium-on-carbon at room temperature and under atmosphericpressure for 1.5 hours to give 0.33 g of N₂-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -tert.butyl-L-prolinamide asa gum which crystallized on standing.

EXAMPLE 93

0.167 g of N² -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide was dissolved in 10 ml of drydimethylformamide and the solution was stirred at 0° C. while 0.191 g ofthe N-(benzyloxycarbonyl)-L-phenylglycine succinimide ester was added asa solid. The solution obtained was stirred at 0° C. for 1 hour and thenstored at 4° C. overnight. The solution was then evaporated in a vacuumand the residue was partitioned between ethyl acetate and water. Theorganic phase was washed with saturated aqueous sodium bicarbonatesolution and then with saturated sodium chloride solution. The aqueousphases were back-extracted with ethyl acetate. The combined organicphases were dried over sodium sulphate and evaporated to give a gumwhich was chromatographed on silica gel using acetone/dichloromethane(1:1) for the elution. The combined product-containing fractions wereevaporated to give a gum which was re-evaporated with diethyl ether togive 0.127 g of a solid. This solid was extracted with dichloromethaneand the combined organic phases were evaporated to give a solid whichwas triturated with diethyl ether. There was thus obtained 0.05 g of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-phenylglycyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a solid of melting point 101°-103° C.

EXAMPLE 94

In a manner analogous to that described in Example 92, from 0.15 g ofN-(benzyloxycarbonyl)-L-phenylalanine, 0.066 g of hydroxybenzotriazole,0.1 g of dicyclohexylcarbodiimide and 0.166 g of N²-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -tert.butyl-L-prolinamidethere was obtained 0.1 g of N²-[3(S)-[N-(benzyloxycarbonyl)-L-phenylalanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide as a white solid of melting point 78°-80° C.

EXAMPLE 95

In a manner analogous to that described in Example 92, from 0.152 g ofN-(benzyloxycarbonyl)-3-cyclohexyl-L-alanine, 0.066 g ofhydroxybenzotriazole, 0.1 g of dicyclohexylcarbodiimide and 0.166 g ofN₂ -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹ -tert.butyl-L-prolinamidethere was obtained 0.1 g of N²-[3(S)-[[N-(benzyloxycarbonyl)-3-cyclohexyl-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide of melting point 71°-75° C.

EXAMPLE 96

In a manner analogous to that described in Example 92, from 0.1 g ofN-(benzyloxycarbonyl)-L-asparagine, 0.05 g of hydroxybenzotriazole,0.078 g of dicyclohexylcarbodiimide and 0.17 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Rand S)-piperazinecarboxamide there was obtained, after trituration withdiethyl ether, 0.11 g of a white solid. This solid was purified by flashchromatography on silica gel using 10% methanol in dichloromethane forthe elution. The first product eluted (isomer A) was 0.043 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide; MS: m/e 697 [M+H]⁺. The second producteluted (isomer B) was 0.007 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide; MS: m/e 697 [M+H]⁺.

The1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Rand S)-piperazinecarboxamide used as the starting material was preparedas follows:

(i) 0.65 g of 2-piperazinecarboxylic acid was dissolved in a mixture of5 ml of water and 5 ml of dioxane, treated with 0.42 g of sodiumbicarbonate and stirred for 5 minutes. 1.09 g of di(-tert.butyl)dicarbonate were added and the mixture was stirred overnight. Themixture was concentrated by evaporation and the residue was extractedexhaustively with ethyl acetate. This procedure was repeated at pH 6 andpH 4. The aqueous layer, pH 4, was then extracted with n-butanol. Thecombined organic extracts were dried over anhydrous sodium sulphate andevaporated to give 0.34 g of4-(tert.butoxycarbonyl)-2-piperazinecarboxylic acid as a cream colouredsolid of melting point 226°-229° C.

(ii) 0.1 g of 4-(tert.butoxycarbonyl)-piperazine-2-carboxylic acid wasdissolved in 10 ml of 1N sodium hydroxide solution, cooled to 0° C. andtreated with 0.2 g of benzyl chloroformate. The mixture was allowed towarm to room temperature and was then stirred overnight. The mixture wasextracted with diethyl ether. The aqueous phase was then acidified to pH4 with 2M hydrochloric acid and extracted with ethyl acetate to give0.06 g of1-(benzyloxycarbonyl)-4-(tert.butoxycarbonyl)-2-piperazinecarboxylicacid as a white solid; MS: m/e 365 [M+H]⁺.

(iii) 0.285 g of1-(benzyloxycarbonyl)-4-(tert.butoxycarbonyl)-2-piperazinecarboxylicacid was dissolved in 10 ml of dry tetrahydrofuran and cooled to -15° C.while stirring. There was then added 0.09 g of N-ethylmorpholinefollowed immediately by 0.107 g of isobutyl chloroformate. The mixturewas stirred for 5 minutes and then 0.2 g of tert.butylamine was addeddropwise. Stirring was continued overnight, during which time themixture was allowed to reach room temperature. The solvent was removedby evaporation and there was obtained a buff coloured oil which waspartitioned between ethyl acetate and water. The organic phase waswashed in sequence with 10% citric acid solution, sodium bicarbonatesolution and saturated sodium chloride solution and dried over anhydroussodium sulphate. The solvent was removed by evaporation to give 0.185 gof1-(benzyloxycarbonyl)-4-(tert.butoxycarbonyl)-N-tert.butyl-2-piperazinecarboxamideas a light brown oil; MS: m/e 420 [M+H]⁺.

(iv) 1.1 g of1-(benzyloxycarbonyl)-4-(tert.butoxycarbonyl)-N-tert.butyl-2-piperazinecarboxamidewere dissolved in 40 ml of ethanol. 0.1 g of 10% palladium-on-carbon wasadded and the mixture was hydrogenated at room temperature and underatmospheric pressure for 2 hours. The catalyst was filtered off. Thefiltrate was evaporated to give 0.74 g of crude product which waspurified by flash chromatography on silica gel using 5% methanol indichloromethane for the elution. After evaporation of the solvents therewas obtained 0.44 g of4-(tert.butoxycarbonyl)-N-tert.butyl-2-piperazinecarboxamide as an oil;MS: m/e 286 [M+H]⁺.

(v) 0.395 g of4-(tert.butoxycarbonyl)-N-tert.butyl-2-piperazinecarboxamide wasdissolved in 50 ml of dry isopropanol and treated with 0.413 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane. The mixture wasstirred at room temperature for 72 hours. The solvent was removed byevaporation to give a brown semi-solid material which was purified byflash chromatography on silica gel using 5% methanol in dichloromethanefor the elution. There was obtained 0.234 g of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide as a mixture of diastereomers.

(vi) 0.234 g of1-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Rand S)-piperazinecarboxamide was dissolved in 20 ml of ethanol andtreated with 100 mg of 10% palladium-on-carbon. The mixture washydrogenated at room temperature and under atmospheric pressure for 2.5hours. The catalyst was filtered off and the filtrate was evaporated togive 0.17 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Rand S)-piperazinecarboxamide which was used without furtherpurification.

EXAMPLE 97

0.035 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide (isomer A) was dissolved in 5 ml of ethylacetate and treated with 5 drops of a saturated solution of hydrogenchloride in ethyl acetate. The mixture was left to stand at roomtemperature for 1 hour and then worked-up to give, afterrecrystallization from ethanol/diethyl ether. 0.024 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2-(Ror S)-piperazinecarboxamide hydrochloride (isomer A) of melting point175°-180° C.

EXAMPLE 98

0.01 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(Ror S)-piperazinecarboxamide (isomer B) was treated with hydrogenchloride in ethyl acetate as described in Example 97 to give 0.007 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(Ror S)-piperazinecarboxamide hydrochloride as a very hygroscopic solid;MS: m/e 597 [M+H]⁺.

EXAMPLE 99

In a manner analogous to that described in Example 92, from 0.091 g ofN-(benzyloxycarbonyl)-3-cyano-L-alanine, 0.05 g of hydroxybenzotriazole.0.076 g of dicyclohexylcarbodiimide and 0.164 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(R,S)-piperazinecarboxamidethere was obtained 0.075 g of a 60:40 mixture of diastereomers of1-[3(S)-[[N-(benzyloxycarbonyl)-3-cyano-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2-piperazinecarboxamideas a buff coloured solid of melting point 80°-85° C.

EXAMPLE 100

In a manner analogous to that described in Example 92, from 0.108 g of3-cyano-N-(2-naphthoyl)-L-alanine, 0.054 g of hydroxybenzotriazole,0.083 g of dicyclohexylcarbodiimide and 0.18 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(R,S)-piperazinecarboxamidethere was obtained 0.015 g of a 1:1:1:1 mixture of isomers of4-(tert.butoxycarbonyl)-N-tert.butyl-1-[3(S)-[[3-cyano-N-(2-naphthoyl)-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-2(R,S)-piperazinecarboxamideas a solid; MS: m/e 699 [M+H]⁺.

The 3-cyano-N-(2-naphthoyl)-L-alanine used as the starting material wasprepared as follows:

0.114 g of 3-cyano-L-alanine was dissolved in 5 ml of 1N sodiumhydroxide solution and treated with 0.285 g of 2-naphthoyl chloride at0° C. After acidification with 2M hydrochloric acid and flashchromatography on silica gel using 25% methanol in dichloromethane forthe elution there was obtained 0.049 g of3-cyano-N-(2-naphthoyl)-L-alanine of melting point 95°-100° C.

EXAMPLE 101

In a manner analogous to that described in Example 92, from 0.048 g of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-4-tert.butyl-2(Rand S)-piperazinecarboxamide, 0.015 g of quinaldic acid, 0.012 g ofhydroxybenzotriazole and 0.018 g of dicyclohexylcarbodiimide there wereobtained, after extensive flash chromatography on silica gel using 10%methanol in dichloromethane for the elution, 0.004 g of a pure singleisomer A,4-(tert.butoxycarbonyl)-N-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2(Ror S)-piperazinecarboxamide, MS: m/e 718 [M+H]⁺, and 0.003 g of a singleisomer B, 4-(tert.butoxycarbonyl)-N-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-2(Ror S)-piperazine carboxamide; MS: m/e 718 [M+H]⁺.

The1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-4-tert.butyl-2(Rand S)-piperazinecarboxamide used as the starting material was preparedas follows:

0.06 g of1-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-N-tert.butyl-2(R,S)-piperazinecarboxamide(i.e. the product of Example 96 prior to the separation of isomers A andB) was dissolved in 20 ml of ethanol. 0.03 g of palladium-on-carbon wasadded and the mixture was hydrogenated at room temperature and underatmospheric pressure for 2 hours. The catalyst was filtered off and thefiltrate was evaporated to give 0.048 g of1-[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4-(tert.butoxycarbonyl)-4-tert.butyl-2-piperazinecarboxamidewhich was used without further purification.

EXAMPLE 102

In a manner analogous to that described in Example 92, from 0.151 g ofN-(benzyloxycarbonyl)-3-cyano-L-alanine, 0.082 g ofhydroxybenzotriazole, 0.126 g of dicyclohexylcarbodiimide and 0.212 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamidethere was obtained, after recrystallization from diethyl ether/n-hexane,0.085 g of1-[3(S)-[[N-(benzyloxycarbonyl)-3-cyano-L-alanyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideas a white solid of melting point 74°-77° C.

EXAMPLE 103

In a manner analogous to that described in Example 92, from 0.372 g ofN-(benzyloxycarbonyl)-L-aspartic acid, 0.189 g of hydroxybenzotriazole,0.288 g of dicyclohexylcarbodiimide and 0.54 g of N²-[3(S)-amino-2(R)-hydroxy-4-(2-naphthyl)butyl]-N¹-tert.butyl-L-prolinamide there was obtained, after recrystallizationfrom isopropanol/n-hexane (1:4), 0.105 g of N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-(2-naphthyl)butyl]-N¹-tert.butyl-L-prolinamide of melting point 149°-151° C.

The N₂ -[3(S)-amino-2(R)-hydroxy-4-(2-naphthyl)butyl]-N¹-tert.butyl-L-prolinamide used as the starting material was prepared asfollows:

(i) 5 g of 3-(2-naphthyl)-L-alanine and 0.93 g of sodium hydroxide in 12ml of water were cooled to 0° C. and stirred while a solution of 1.4 gof sodium hydroxide in 9 ml of water and 5 ml of benzyl chloroformatewere added simultaneously during 10 minutes. Stirring was continued for2 hours and the mixture was allowed to come to room temperature. Themixture was diluted with water and then extracted with diethyl ether.The aqueous layer was acidified with 4 ml of concentrated hydrochloricacid and extracted with ethyl acetate. The ethyl acetate extracts wereback-washed with water. The combined ethyl acetate extracts were driedover sodium sulphate, filtered, evaporated and triturated with petroleumether (boiling point 40°-60° C.) to give 7.5 g ofN-(benzyloxycarbonyl)-3-(2-naphthyl)-L-alanine of melting point109°-111° C.

(ii) A solution of 7.5 g ofN-(benzyloxycarbonyl)-3-(2-naphthyl)-L-alanine in 20 ml of drytetrahydrofuran was stirred at -8° C. and treated with 3.5 ml ofN-ethylmorpholine followed by 4 ml of isobutyl chloroformate, addeddropwise over a period of 10 minutes. The mixture was stirred at -8° C.for a further 20 minutes and cold (0° C.) anhydrous diethyl ether wasadded. The resulting white precipitate was filtered off and the coldfiltrate was added dropwise to 100 ml of a stirred, cold (-8° C.)solution of diazomethane in diethyl ether. The cooling was removed andthe solution was stirred for 3 hours. Water was added while stirring.The organic phase was washed in sequence with water, sodium bicarbonatesolution and saturated sodium chloride solution and then dried oversodium sulphate. The solvent was removed by evaporation to give a yellowoil which, on evaporation with petroleum ether (boiling point 40°-60°C.), gave 9.4 g of a solid. Trituration of this solid with diethyl etherand refrigeration at 4° C. overnight gave 3.2 g of benzyl[3-diazo-1(S)-[(2-naphthyl)methyl]-2-oxopropyl] carbamate.

(iii) 3.13 g of benzyl[3-diazo-1(S)-[(2-naphthyl)methyl]-2-oxopropyl]carbamate were dissolvedin 200 ml of anhydrous diethyl ether and stirred while hydrogen chloridegas was bubbled through the solution. After 1 hour theinitially-precipitated solid became more granular and excess hydrogenchloride was being emitted. Then, the solvent was removed by evaporationat room temperature and the resulting white solid was dried and freedfrom entrained hydrogen chloride in a vacuum over sodium hydroxide for 2hours. The resulting benzyl[3-chloro-1(S)-[(2-naphthyl)methyl]-2-oxopropyl] carbamate was usedimmediately in the next step.

(iv) The foregoing benzyl[3-chloro-1(S)-[(2-naphthyl)methyl]-2-oxopropyl] carbamate was dissolvedin 100 ml of 10% aqueous tetrahydrofuran, cooled to 0° C. and treatedwith 0.456 g of sodium borohydride which was added carefully as thesolid. The mixture was stirred at 0° C. for 1 hour and then at roomtemperature overnight. The mixture was evaporated to give a white solidwhich was partitioned between dichloromethane and water. The stirredmixture was carefully acidified to pH 1 with concentrated hydrochloricacid. The phases were separated and the aqueous phase was back-extractedwith dichloromethane. The combined organic phases were dried overanhydrous sodium sulphate and evaporated to give 4.325 g of crudeproduct of melting point 155°-160° C. This crude product was extractedwith boiling n-hexane. After removal of the solvent by evaporation theresidue was recrystallized from ethyl acetate/n-hexane to give 1.046 gof pure benzyl [3-chloro-2(S)-hydroxy-1(S)-[(2-naphthyl)methyl]propyl]carbamate of melting point 173°-174° C.

(v) 1.02 g of benzyl[3-chloro-2(S)-hydroxypropyl-1(S)-[(2-naphthyl)methyl]propyl] carbamatewere stirred in 40 ml of ethanol with 4 ml of 0.7M potassium hydroxidesolution in ethanol for 0.75 hours. The solvent was removed byevaporation and the residue was partitioned between dichloromethane andwater. The organic phase was dried over anhydrous sodium sulphate andevaporated to give a white solid which was recrystallized from ethylacetate/n-hexane. There was obtained 0.879 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-(2-naphthyl)butane as a whitesolid of melting point 115°-116° C.

(vi) 0.465 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-(2-naphthyl)butane and 0.251 gof L-proline tert.butylamide in 10 ml of dry isopropanol was heated at80° C. for 23 hours and worked-up as described in Example 92(i) to give0.483 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-(2-naphthyl)butyl]-N¹-tert.butyl-L-prolinamide as a white foam melting at about 75°-85° C.

(vii) A solution of 0.725 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-(2-naphthyl)butyl]-N¹-tert.butyl-L-prolinamide was dissolved in 25 ml of ethanol andhydrogenated at room temperature and under atmospheric pressure over 0.5g of 10% palladium-on-carbon for 20 hours. The catalyst was filtered offand the filtrate was evaporated to give 0.54 g of N₂-[3(S)-amino-2(R)-hydroxy-L-(2-naphthyl)butyl]-N¹-tert.butyl-L-prolinamide as a white foam which was used without furtherpurification.

EXAMPLE 104

In a manner analogous to that described in Example 92, from 0.133 g ofN-(benzyloxycarbonyl)-L-asparagine, 0.068 g of hydroxybenzotriazole,0.103 g of dicyclohexylcarbodiimide and 0.16 g of N₂-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide, but using dichloromethane inplace of ethyl acetate as the partitioning solvent and 20% methanol indichloromethane for the chromatography and carrying out there-evaporation with diethyl ether, there was obtained 0.1 g of N₂-[3(S)-[N-(benzyloxycarbonyl)-L-asparaginylamino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide of melting point 115° C.

The N₂ -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide used as the starting material wasprepared as follows:

(i) 2.65 g of N-(benzyloxycarbonyl)-4(R)-hydroxy-L-proline weredissolved in 10 ml of dry tetrahydrofuran and cooled to -10° C. whilestirring with a magnetic stirrer. 1.15 g of N-ethylmorpholine were addedfollowed immediately by 1.36 g of isobutyl chloroformate. The mixturewas stirred at -10° C. for 30 minutes and then 2.19 g of tert.butylaminewere added. Stirring was continued at -10° C. for 1 hour, the mixturewas allowed to warm to room temperature during 2 hours and was then leftto stand for 2 hours. The solvent was removed by evaporation in a vacuumand the residue was partitioned between ethyl acetate and water. Theorganic layer was washed with 10% citric acid solution and sodiumbicarbonate solution and then dried over sodium sulphate. The solventwas removed by evaporation to give a solid which was triturated withdiethyl ether and filtered off. There were obtained 2.23 g of crudeproduct which was recrystallized from ethyl acetate/diethyl ether togive 1.57 g of N₂ -(benzyloxycarbonyl)-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide of melting point 128°-130° C.

(ii) 0.224 g of N₂ -(benzyloxycarbonyl)-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide was dissolved in 20 ml of ethanoland hydrogenated over 50 mg of 10% palladium-on-carbon at roomtemperature and under atmospheric pressure for 2 hours. The catalyst wasfiltered off and the filtrate was evaporated to give 0.13 g of N¹-tert.butyl-4(R)-hydroxy-L-prolinamide which was used in the next stepwithout further purification.

(iii) 0.13 g of N¹ -tert.butyl-4(R)-hydroxy-L-prolinamide and 0.208 g of3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in 10 ml of dryisopropanol were heated at 80° C. for 24 hours. Working-up in a manneranalogous to that described in Example 33(iii) gave, after triturationwith diethyl ether, 0.236 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide as a white gelatinous solid ofmelting point 135° C.

(iv) 0.226 g of N²-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide was dissolved in 20 ml of ethanoland hydrogenated over 40 mg of 10% palladium-on-carbon at roomtemperature and under atmospheric pressure for 2 hours. The catalyst wasremoved by filtration and the filtrate was evaporated to give 0.16 g ofN₂ -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(R)-hydroxy-L-prolinamide as a gum which was used withoutfurther purification.

EXAMPLE 105

In a manner analogous to that described in Example 92, but usingdichloromethane in place of ethyl acetate as the partitioning solvent,using 20% methanol in dichloromethane for the chromatography andcarrying out the re-evaporation with diethyl ether, from 0.067 g ofN-(benzyloxycarbonyl)-L-asparagine, 0.034 g of hydroxybenzotriazole,0.052 g of dicyclohexylcarbodiimide and 0.08 g of N²-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide there was obtained 0.03 g of N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide as a cream coloured solid meltingat about 140° C.

The N₂ -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide used as the starting material wasprepared as follows:

(i) 1.7 g of N-(benzyloxycarbonyl)-4(S)-hydroxy-L-proline were dissolvedin 15 ml of dry dimethylformamide while stirring-and the solution wascooled to 0° C. 0.817 g of N-hydroxysuccinimide was added and themixture was stirred while the temperature was allowed to rise to 20° C.The mixture was then stirred overnight at room temperature. Theresulting dicyclohexylurea was filtered off and the filtrate was cooledto -10° C. 2 ml of tert.butylamine were then added while stirring.Stirring was continued while the mixture was allowed to warm to roomtemperature and the mixture was then stirred overnight. The separatedsolid was filtered off and the filtrate was evaporated in a vacuum togive a gum which was partitioned between ethyl acetate and water. Theorganic layer was washed with 10% citric acid solution and then withsaturated sodium bicarbonate solution. The aqueous phases wereback-extracted twice with ethyl acetate. The combined organic phaseswere dried over anhydrous sodium sulphate and evaporated to give a solidwhich was purified by flash chromatography on silica gel using 5%methanol in dichloromethane for the elution. There were obtained 1.36 gof a crude product which was recrystallized from ethyl acetate/diethylether/petroleum ether (boiling point 40°-60° C.) (1:4:4) and then storedat 4° C. in a refrigerator overnight. There were thus obtained 1.127 gof N₂ -(benzyloxycarbonyl)-N¹ -tert.butyl-4(S)-hydroxy-L-prolinamide ofmelting point 131°-132° C.

(ii) 0.224 g of N₂ -(benzyloxycarbonyl)-N¹-tert-butyl-4(S)-hydroxy-L-prolinamide was hydrogenated in a manneranalogous to that described in Example 104 (ii) to give 0.135 g of N¹-tert.butyl-4(S)-hydroxy-L-prolinamide as a gum-like solid which wasused in the next step without purification.

(iii) 0.135 g of N¹ -tert.butyl-4(S)-hydroxy-L-prolinamide and 0.208 gof 3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in 10 ml of dryethanol was stirred for 4 days at room temperature. The mixture wasworked-up as described in Example 92(i), with the exception that thechromatography was carried out using 10% methanol in dichloromethane forthe elution. There was obtained 0.11 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide as a foam.

(iv) 0.11 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide was dissolved in 10 ml of ethanoland hydrogenated over 20 mg of 10% palladium-on-carbon at roomtemperature and under atmospheric pressure for 2 hours. The catalyst wasfiltered off and the filtrate was evaporated to give 0.08 g of N₂-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide as a gum which was used withoutfurther purification.

EXAMPLE 106

In a manner analogous to that described in Example 92, but usingdichloromethane in place of ethyl acetate as the partitioning solvent,using 20% methanol in dichloromethane for the chromatography andcarrying out the re-evaporation-with diethyl ether, from 0.105 g ofN-(benzyloxycarbonyl)-L-asparagine, 0.054 g of hydroxybenzotriazole,0.082 g of dicyclohexylcarbodiimide and 0.17 g of N₂-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4(R)-(tert.butoxyformamido)-N.sup.1-tert.butyl-L-prolinamide there was obtained 0.045 g of N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide as an off-white solid of melting point170°-175° C.

The N₂ -[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4(R)-N¹-(tert.butoxyformamido)-N¹ -tert.butyl-L-prolinamide used as thestarting material was prepared as follows:

(i) 0.32 g of N₂ -(benzyloxycarbonyl)-N¹-tert.butyl-4(S)-hydroxy-L-prolinamide was dissolved in 5 ml of drypyridine while stirring, cooled to 0° C. and treated dropwise with 0.82ml of methanesulphonyl chloride. The solution was stirred at 0° C. for afurther 2 hours. The mixture was poured into a mixture of ice and waterwhich was then extracted with ethyl acetate. The combined organicextracts were washed with 2M hydrochloric acid and then with saturatedsodium bicarbonate solution and subsequently dried over anhydrous sodiumsulphate. After evaporation there was obtained 0.5 g of N²-(benzyloxycarbonyl)-N¹-tert.butyl-4(S)-(methanesulphonyloxy)-L-prolinamide as an oil which wasused without further purification.

(ii) 0.5 g of N₂ -(benzyloxycarbonyl)-N¹-tert.butyl-4(S)-(methanesulphonyloxy)-L-prolinamide was dissolved in 10ml of dry dimethylformamide and treated with 0.330 g of sodium azide.The heterogeneous mixture was stirred and heated at 75° C. for 18 hours.The mixture was evaporated under an oil pump vacuum to give a solidwhich was partitioned between ethyl acetate and water. The ethyl acetatephase was washed with saturated sodium chloride solution and dried overanhydrous sodium sulphate. The solvent was removed by evaporation togive 0.345 g of N₂ -(benzyloxycarbonyl)-4(R)-azido-N¹-tert.butyl-L-prolinamide in the form of a gum.

(iii) 0.345 g of N² -(benzyloxycarbonyl)-4(R)-azido-N¹-tert.butyl-L-prolinamide was dissolved in 5 ml of dry tetrahydrofuranand the solution was evaporated. The residual gum was dissolved in 10 mlof dry tetrahydrofuran and treated under a nitrogen atmosphere with0.262 g of triphenylphosphine. The resulting solution was left to standunder a nitrogen atmosphere at room temperature for 18 hours. 0.027 g ofwater was added and the solution was left to stand at room temperaturefor 24 hours. The solvent was then removed by evaporation and theresidual gum was partitioned between water and diethyl ether. Theaqueous phase was back-extracted with diethyl ether. The aqueous phasewas then evaporated to give 0.09 g of N₂-(benzyloxycarbonyl)-4(R)-amino-N¹ -tert-butyl-L-prolinamide as a gum.After standing overnight at room temperature the diethyl ether extractswere combined and evaporated to give 0.7 g of an oil which waschromatographed on silica gel using 10% methanol in dichloromethane forthe elution to give a further 0.16 g of N²-benzyloxycarbonyl)-4(R)-amino-N¹ -tert.butyl-L-prolinamide as a gum.

(iv) 0.21 g of N² -(benzyloxycarbonyl)-4(R)-amino-N¹-tert.butyl-L-prolinamide was dissolved in a mixture of 5 ml of dioxaneand 5 ml of water. 0.056 g of sodium bicarbonate was added to give asolution to which 0.144 g of di(tert.butyl) dicarbonate was added. Themixture obtained was stirred at room temperature overnight. Solventswere removed by evaporation and the residue was partitioned betweenwater and diethyl ether. The aqueous phase was back-extracted withdiethyl ether and then with ethyl acetate. The combined organic extractswere washed with saturated sodium chloride solution and dried overanhydrous sodium sulphate. The dried extracts were combined andevaporated to give 0.27 g of N²-(benzyloxycarbonyl)-4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide as an off-white foam.

(v) 0.25 g of N₂ -(benzyloxycarbonyl)-4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide was dissolved in ethanol and hydrogenated over0.1 g of 10% palladium-on-carbon at room temperature and underatmospheric pressure for 4 hours. The catalyst was filtered off and thefiltrate-was evaporated to give 0.17 g of 4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide as a glass which was used without furtherpurification.

(vi) 0.17 g of 4(R)-(tert.butoxyformamido)-N¹ -tert.butyl-L-prolinamideand 0.178 g of 3(S)-(benzyloxyformamido)-1,2(S)-epoxy-4-phenylbutane in15 ml of dry ethanol were stirred and the resulting solution was left tostand at room temperature for 4.5 days. The solution was then heated at40° C. for 96 hours. Working-up as described in Example 92(i), with theexception that the chromatography was carried out using 10% methanol indichloromethane for the elution, gave 0.230 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenyl]butyl]-4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide as a gum.

(vii) 0.22 g of N₂-[3(S)-(benzyloxyformamido)-2(R)-hydroxy-4-phenylbutyl]-4(R)-(tert.butoxyformamido)-N¹-tert.butyl-L-prolinamide was dissolved in 10 ml of ethanol andhydrogenated over 0.05 g of 10% palladium-on-carbon at room temperatureand under atmospheric pressure for 2 hours. The catalyst was filteredoff and the filtrate was evaporated to give 0.17 g of N₂-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-4(R)-(tert.butoxyformamido)-N.sup.1-tert.butyl-L-prolinamide as a gum which was used without furtherpurification.

EXAMPLE 107

In a manner analogous to that described in Example 27, from 162 mg of2-[[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenyl]butyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1(Ror S)-carboxamide, (isomer B), 55 mg of quinaldic acid, 43 mg of1-hydroxybenzotriazole, 0.04 ml of N-ethylmorpholine and 66 mg ofdicyclohexylcarbodiimide there were obtained, after chromatography onsilica gel using 3% methanol in ethyl acetate for the elution, 95 mg ofN-tert.butyl-1,2,3,4-tetrahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]pyrido[3,4-b]indole-1(Ror S)-carboxamide; MS: m/e 707 [M+H]⁺.

The2-[[3(S)-[[L-asparaginyl]amino]-2(R)-hydroxy-4-phenyl]butyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1(Ror S)-carboxamide used as the starting material was prepared byhydrogenating2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-1(Ror S)-carboxamide.

EXAMPLE 108

A solution of 154 mg oftrans-2-[3(S)-[(L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamideand 52 mg of quinaldic acid in 6 ml of dry tetrahydrofuran is cooled inan ice/salt mixture. 41 mg of hydroxybenzotriazole, 35 mg=ofN-ethylmorpholine and 68 mg of dicyclohexylcarbodiimide are added andthe mixture is stirred for 64 hours. The mixture is diluted with ethylacetate and filtered. The filtrate is washed with aqueous sodiumbicarbonate solution and with sodium chloride solution and thenevaporated. The residue is chromatographed on silica gel usingdichloromethane/methanol (9:1) for the elution to givetrans-N-tert.butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(4aR,8aS)-isoquinoline-3(S)-carboxamide.

Thetrans-2-[3(S)-[(L-asparaginyl)amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamideto be used as the starting material is prepared by hydrogenatingtrans-2-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-decahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamide.

EXAMPLE 109

A solution of 1.02 g of1-[3(S)-amino-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamideand 685 mg of N-(tert.butoxycarbonyl)-S-methyl-L-cysteine in 7 ml of drytetrahydrofuran was cooled in an ice/salt mixture. 394 mg ofhydroxybenzotriazole, 335 mg of N-ethylmorpholine and 661 mg ofdicyclohexylcarbodiimide were added and the mixture was stirred for 3hours. The mixture was diluted with ethyl acetate and filtered. Thefiltrate was washed with aqueous sodium bicarbonate solution and withsodium chloride solution and then evaporated. The solvent was removed byevaporation and the residue was chromatographed on silica gel usingdichloromethane/methanol (96:4) for the elution to give 630 mg of1-[3(S)-[[N-(tert.butoxycarbonyl)-L-cysteinyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamidein the form of a white solid; MS: m/e 565 [M+H]⁺.

EXAMPLE 110

A solution of 650 mg ofN-tert.butyl-1-[3(S)-[(L-cysteinyl)amino]-2(R)-hydroxy-4-phenylbutyl]-2(S)-piperidinecarboxamideand 242 mg of quinaldic acid was cooled in an ice/salt mixture. 189 mgof hydroxybenzotriazole, 161 mg of N-ethylmorpholine and 317 mg ofdicyclohexylcarbodiimide were added and the mixture was stirred for 64hours. The mixture was diluted with ethyl acetate and filtered, and thefiltrate was then evaporated. The residue was partitioned betweendichloromethane and aqueous sodium bicarbonate solution. The organicphase was washed with sodium chloride solution and then evaporated. Theresidue was chromatographed on silica gel using dichloromethane/methanol(19:1) for the elution to give 350 mg ofN-tert.butyl-1-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-cysteinyl]amino]butyl]-2(S)-piperidinecarboxamideas a white solid; MS: m/e 620 [M+H]⁺.

TheN-tert.butyl-1-[3(S)-[(L-cysteinyl)amino]-2(R)-hydroxy-4-phenylbutyl]-2(S)-piperidinecarboxamideused as the starting material was prepared as follows:

A solution of 930 mg of1-[3(S)-[[N-(tert.butoxycarbonyl)-L-cysteinyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N-tert.butyl-2(S)-piperidinecarboxamidein 7 ml of trifluoroacetic acid was stirred at 20° C. for 1 hour. Themixture was then evaporated to dryness and the residue was partitionedbetween dichloromethane and aqueous sodium bicarbonate solution. Theorganic phase was evaporated to give 650 mg ofN-tert.butyl-1-[3(S)-[(L-cysteinyl)amino]-2(R)-hydroxy-4-phenylbutyl]-2(S)-piperidinecarboxamideas a colourless gum; MS: m/e [M+H]⁺.

EXAMPLE 111

15 mg of freshly distilled acetyl chloride were added to a solution,which was cooled to 0° C. and stirred, of 12 mg of 4(R)-amino-N²-[3(S)-[[N-benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenyl-butyl]-N¹-tert.butyl-L-prolinamide hydrochloride and 21 mg of sodium bicarbonatein 0.5 ml of water and 0.25 ml of dimethylformamide. The mixture wasstirred vigorously at 0° C. for 5 hours and then left to stand at roomtemperature overnight. The mixture was diluted with water and extractedwith dichloromethane. The combined dichloromethane extracts wereevaporated to give a gum which was purified by flash chromatography onsilica gel using 20% methanol in dichloromethane for the elution. Therewere obtained 2 mg of 4(R)-acetylamino-N₂-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert-butyl-L-prolinamide as a semi-solid; MS: m/e 639 [M+H]⁺.

The 4(R)-amino-N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide hydrochloride used as the starting materialwas prepared as follows:

29 mg of N₂ -[3(S)-[[N₂-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-4(R)-(tert-butoxyformamido)-N¹-tert.butyl-L-prolinamide were dissolved in 0.5 ml of a saturatedsolution of hydrogen chloride in ethyl acetate and left to stand at roomtemperature for 1 hour. The solution was evaporated and the residue wastriturated with diethyl ether and stored at 4° C. overnight. Theseparated solid was filtered off and washed with diethyl ether to give19 mg of 4(R)-amino-N²-[3(S)-[[N-(benzyloxycarbonyl)-L-asparaginyl]amino]-2(R)-hydroxy-4-phenylbutyl]-N¹-tert.butyl-L-prolinamide hydrochloride as a solid of melting point206°-210° C.

The following Example illustrates the manufacture of a pharmaceuticalpreparation containing a compound of formula I or a pharmaceuticallyacceptable acid addition salt thereof as the active ingredient:

EXAMPLE A

An aqueous solution of the active ingredient is filtered sterile andmixed while warming with a sterile gelatine solution, which containsphenol as a preserving agent, using amounts such that 1.00 ml of theresulting solution contains 3.0 mg of active ingredient, 150.0 mg ofgelatine, 4.7 mg of phenol and distilled water ad 1.0 ml. The mixture isfilled into vials of 1.0 ml capacity under aseptic conditions.

We claim:
 1. A compound of the formula ##STR15## wherein n is zero or 1;R¹ is cycloalkylcarbonyl, aralkanoyl, and aroyl; R² is hydrogen; R³ isalkyl, cycloalkyl, aryl, aralkyl, cyanoalkyl, alkylsulphinylalkyl,carbamoylalkyl or alkoxycarbonylalkyl or, when n is zero, R³ isadditionally alkylthioalkyl or, when n is 1, R³ can also bealkylsulphonylalkyl; R⁴ is alkyl, cycloalkyl, cycloalkylalkyl, aryl oraralkyl; R⁵ is hydrogen and R⁶ is hydroxy or R⁵ and R⁶ together are oxo;--N(R⁷)--CH(R⁸)(R⁹) is selected from the group consisting of ##STR16##where m is 2 and p is 1 or 2, R⁹ is alkoxycarbonyl, monoalkylcarbamoyl,monoaralkylcarbamoyl, monoarylcarbamoyl or a group of the formula##STR17## where R¹⁰ and R¹¹ are alkyl, or the pharmaceuticallyacceptable addition salts thereof.
 2. The compound of claim 1 wherein nis zero.
 3. The compound of claim 2 wherein R³ is alkyl, cycloalkyl,aryl, aralkyl, cyanoalkyl, alkylthioalkyl, carbamoylalkyl oralkoxycarbonylalkyl.
 4. The compound of claim 3 wherein R¹ is2-naphthoyl, 1-hydroxy-2-naphthoyl, 3-hydroxy-2-naphthoyl or3-benzyloxy-2-naphthoyl, and R² is hydrogen.
 5. The compound of claim 4wherein R³ is cyanomethyl, methylthiomethyl or carbamoylmethyl, R⁴ isbenzyl, and R⁵ is hydrogen.
 6. The compound of claim 5 wherein R⁹ isselected from tert.butoxycarbonyl, isobutylcarbamoyl,tert.butylcarbamoyl or ##STR18## wherein R¹⁰ is sec.butyl and R¹¹ isisobutyl.
 7. The compound of claim 1 wherein R³ is heterocyclylalkyl. 8.A method for the treatment of viral infections, comprisingadministration of an antivirally effective amount of the compound offormula I in claim
 1. 9. A method for the treatment of viral infections,comprising administration of an antivirally effective amount of thecompound of formula I in claim
 7. 10. A pharmaceutical compositioncomprising a compound of formula I of claim 1, and one or moretherapeutically inert exipients.
 11. A pharmaceutical compositioncomprising a compound of formula I of claim 7, one or moretherapeutically inert exipients.